CN107384834A - The application of spindle lysine Bacillus strain, enzyme preparation and its degrading pesticide residues - Google Patents
The application of spindle lysine Bacillus strain, enzyme preparation and its degrading pesticide residues Download PDFInfo
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- CN107384834A CN107384834A CN201710757505.6A CN201710757505A CN107384834A CN 107384834 A CN107384834 A CN 107384834A CN 201710757505 A CN201710757505 A CN 201710757505A CN 107384834 A CN107384834 A CN 107384834A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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Abstract
The invention discloses the application of spindle lysine Bacillus strain, enzyme preparation and its degrading pesticide residues.The invention provides the bacterial strains of spindle lysine bacillus (Lysinibacillus fusiformis) FY 7, its deposit number is CGMCC No.13523.Present invention also offers the screening of bacterial strain, induction, domestication and agricultural chemicals pesticide residual degradation organized enzyme preparation method, i.e., carries out purpose bacterium screening by carbon source of chlopyrifos, and purpose bacterium is carried out into inducing and acclimating in single agricultural chemicals and blended.Bacterial strain is fermented, collects thalline, breaks born of the same parents, centrifugation, supernatant can prepare pesticide residual degradation enzyme preparation, available for the degraded of vegetables and fruits residues of pesticides, have application potential at the degraded residual aspect of vegetables and fruits agriculture.
Description
Technical field
The present invention relates to biotechnology degrading pesticide residues field, more particularly to spindle lysine bacillus
(Lysinibacillus fusiformis) bacterial strain screening, inducing and acclimating and the application in removal vegetables and fruits Pesticide Residues.
Background technology
As the kind of agricultural chemicals, yield and usage amount increase severely in recent years, mixing is using a variety of agricultures in agricultural production process
The phenomenon of medicine happens occasionally and more seriously, causes Multiple Pesticides to appear in identical product.The residual exceeded vegetables water of edible agriculture
Fruit can endanger nerve center, make Nerve conduction disorderly；Can Mutation induction, cause deformity, influence offspring health；Liver can be induced
The change of dirty enzyme, liver enlargement is made so that necrosis；Kidney can be invaded and cause lesion.Remains of pesticide is accumulated in human body, exceedes
Some chronic diseases can be caused after certain limit, such as muscle is numb, cough, or even can induce vascular diseases, diabetes and cancer
Disease etc..
Bioremediation technology has occurred since being the eighties and the removing developed and the biotechnology curbed environmental pollution,
It mainly using the ability of biospecific decomposition poisonous and harmful substance, the pollutant gone in depollution environment such as soil, reaches
Remove the purpose of environmental pollution.Microorganism has stronger adaptation and the ability tamed, by certain adaptation process, micro- life
Thing produces corresponding enzyme system, or by the new enzyme system of the foundation such as gene mutation come new agricultural chemicals of degrading.
Food security is improved to be significant, but at present, there is not yet utilizing bacterial strain or the enzyme preparation being made from it
The report of blended residual in degraded vegetables and fruits.
The content of the invention
It is an object of the present invention to provide the one plant of spindle lysine gemma obtained through screening, agricultural chemicals induction, domestication
Bacillus strain.
Spindle lysine bacillus (Lysinibacillus fusiformis) bacterial strain provided by the invention (is designated as
FY-7), its deposit number is CGMCC No.13523.
Spindle lysine bacillus (Lysinibacillus fusiformis) bacterial strain is through screening purpose bacterium
Strain and to purpose bacterial strain through single agricultural chemicals and blended induction, domestication obtain, comprise the following steps：
1) by the purpose bacterial strain rejuvenation of screening；
2) culture medium containing different active ingredient concentration agricultural chemicals is prepared using agricultural chemicals chlopyrifos, by the purpose of step 1) rejuvenation
Bacterial strain streak inoculation, culture and by continuous induction culturing (by improving constantly pesticide concentration), cultivate be resistant to it is higher
Chlopyrifos concentration aimed strain；
3) agricultural chemicals chlopyrifos, carbendazim, procymidone and dimethomorph isoconcentration are mixed, and after being diluted to finite concentration
It is added in culture medium, is configured to the culture medium containing different effective ingredient concentration agricultural chemicals.The aimed strain that step 2) is obtained again
Streak inoculation, culture simultaneously obtain being resistant to higher agriculture by continuous induction culturing (by improving constantly pesticide concentration), cultivation
The bacterial strain of concentration, final separation obtain one plant of spindle lysine bacillus (Lysinibacillus fusiformis)
Bacterial strain (is designated as FY-7).
Second object of the present invention is to provide a kind of method for producing biodegradable enzyme.
The method of production biodegradable enzyme provided by the invention, comprises the following steps：By above-mentioned spindle lysine gemma
After bacillus (Lysinibacillus fusiformis) bacterial strain FY-7 fermentations, thalline, broken born of the same parents, centrifugation are collected, extracts supernatant,
Obtain biodegradable enzyme solutions.
In the method for above-mentioned production biodegradable enzyme, the condition of the fermentation is to be cultivated in 34-39 DEG C, 180-200rpm
16-24 hours.
In the method for above-mentioned production biodegradable enzyme, the component for the fermentation medium that the fermentation uses is as follows：Every liter institute
Stating fermentation medium includes tryptone 17-19g, phytone 1-4g, sodium chloride 2-5g, dipotassium hydrogen phosphate 2-5g and grape
Sugared 2-5g, volume is supplied with water；The pH value of fermentation medium is 6.8-7.6, preferably 7.0-7.4.
In the method for above-mentioned production biodegradable enzyme, the fermentation specifically includes following steps：By spindle lysine bud
Spore bacillus (Lysinibacillus fusiformis) bacterial strain FY-7 strains obtain seed by expanding culture, and seed is inoculated with
Cultivated into the fermentation medium under conditions of the fermentation.
The condition for expanding culture is to cultivate 16-24h in 34-39 DEG C, 180-200rpm.
The strain is by above-mentioned spindle lysine bacillus (Lysinibacillus fusiformis) bacterial strain
FY-7 thalline is suspended in the solution obtained in phosphate buffer.
In the method for above-mentioned production biodegradable enzyme, after the fermentation, by tunning in 12000-14000rpm, temperature
4-6 DEG C of degree, 15-25min is centrifuged, collect thalline, height crushes born of the same parents after thalline is resuspended, then in 8000-10000rpm, temperature 4-
6 DEG C, 50-65min is centrifuged, supernatant is collected, by supernatant liquid filtering, obtains biodegradable enzyme solutions.
Third object of the present invention is to provide a kind of biodegradable enzyme preparation.
Biodegradable enzyme preparation provided by the invention, its active component are above-mentioned spindle lysine bacillus
(Lysinibacillus fusiformis) bacterial strain FY-7 or above-mentioned biodegradable enzymes.
Fourth object of the present invention is to provide above-mentioned spindle lysine bacillus (Lysinibacillus
Fusiformis) bacterial strain FY-7, biodegradable enzyme and biodegradable enzyme preparation are in pesticide residual degradation, particularly vegetables and fruits residues of pesticides
Application in degraded.
The concentration of the biodegradable enzyme or biodegradable enzyme preparation in vegetables and fruits chemical residual degradation is 2.5-
15ppm, optimal is 2.5-7.5ppm, different because of specific vegetables and fruits.
Beneficial effects of the present invention are embodied in：
The experiment proves that present invention induction, domestication obtain spindle lysine bacillus
(Lysinibacillus fusiformis) bacterial strain FY-7, extraction endocellular enzyme can be used as biology drop after fermented and cultured is carried out to it
Enzyme is solved, the residues of pesticides that the biodegradable enzyme may act in vegetables and fruits, makes Multiple Pesticides residue degrading, reaches removal residues of pesticides
Effect, there is application potential in chemical residual degradation field.
Brief description of the drawings
Fig. 1 is inducing and acclimating spindle lysine bacillus (Lysinibacillus fusiformis) bacterial strain FY-7
Original bacteria and inducing and acclimating bacterial strain FY-7 degrading pesticide contrast.
Fig. 2 is the liquid based on spindle lysine bacillus (Lysinibacillus fusiformis) bacterial strain FY-7
The result of the residues of pesticides of state biodegradation enzyme preparation processing cucumber.
Fig. 3 is the liquid based on spindle lysine bacillus (Lysinibacillus fusiformis) bacterial strain FY-7
The result of the residues of pesticides of state biodegradation enzyme preparation processing Lettuce.
Embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.Embodiment facilitates a better understanding of this hair
It is bright, but do not limit the present invention.Test material used, is routine biochemistry reagent unless otherwise specified in embodiment.Implement
% in example, is weight/mass percentage composition unless otherwise specified.Quantitative test in embodiment, it is respectively provided with and repeats to test three times,
Results averaged.Rotating speed in embodiment, it is the rotating speed centrifuged under radius in radius for 5.5cm.
Colorimetric method for determining bacterial number, i.e. turbidity counting method are used in embodiment；With the light absorption value OD of culture600Table
Show the increment of bacterium.
Example 1, the separation identification of spindle lysine Bacillus strain
First, the collection of pedotheque
It is collected in insecticide factory's floss hole sludge.
2nd, the separation screening of bacterial strain
Sludge 10g is weighed in 100mL inorganic salt liquid culture mediums, inorganic salt liquid medium component is as follows：
MgSO4·7H2O 0.2g；K2HPO40.1g；(NH4)2SO40.1g；CaSO40.04g；FeSO4·7H2O
0.001g；Deionized water 1L；pH 7.0.121 DEG C of sterilizing 30min.
The chlopyrifos that concentration is 100mg/L is added in inorganic salt liquid culture medium, nutrient solution is obtained, in 37 DEG C, 180r/
Min shaking table cultures, it is so anti-in 10% inoculum concentration access fresh medium, to transfer weekly once later after cultivating one week
Multiple repeatedly domestication.Finally, isolated and purified, be inoculated into the LB culture mediums containing 100ppm chlopyrifos with plate dilution method,
Select and grow best inoculation inclined-plane, numbering, which preserves, to be used to tame.
3rd, identify
Specific authentication step is as follows：Extraction numbering preserves the STb gene of bacterial strain and is used as template, using universal primer ITS1
(5 '-TCCGTAGGTGAACCTGCGG-3 ') and ITS4 (5 '-TCCTCCGCTTATTGATATG-3 '), PCR expand to obtain it
ITS, 947bp nucleotide sequence (referring to SEQ.ID.NO.1), sequence analysis analysis shows are obtained through sequencing：Numbering
Preserve bacterial strain and the homology highest of spindle lysine bacillus (Lysinibacillus fusiformis).
Example 2, the induction of spindle lysine Bacillus strain, domestication
First, chlopyrifos induction, domestication
1. the active constituent content section that chlopyrifos is prepared：300mg/L-800mg/L.Prepare the chlopyrifos containing various concentrations
Tryptone agar.Different amounts of chlopyrifos is added in tryptone agar respectively the culture medium containing chlopyrifos is made.
Specific steps：1) chlopyrifos is configured to 4500mg/L pesticide standard liquid.The pesticide standard liquid of 4500mg/L concentration is taken respectively
13.3mL, 17.8mL, 22.2mL, 26.6mL, 31.1mL, 35.6mL are added in 200mL tryptone agars, are configured to contain and are poisoned with poison
Tick 300mg/L, 400mg/L, 500mg/L, 600mg/L, 700mg/L and 800mg/L tryptone agar culture medium.Flat board system
Make：By the tryptone agar pour plate of the chlopyrifos containing 300mg/L-800mg/L prepared, 15mL/ wares, solidification to be cooled
After use.
Tryptone agar composition is：Per L tryptones containing 18g, 2g phytones, 5g sodium chloride and 15g agar
(pH is 7.3 ± 0.2).
2. all purposes bacterial strain is distinguished into streak inoculation in middle 300mg/L flat board.
3. the flat board being inoculated with is placed in 37 DEG C of constant incubators, 24h is cultivated.
4. then by the flat board of cultured bacterial strain renewed vaccination to 400mg/L.The flat board being inoculated with is placed in 37 DEG C of perseverances
In warm incubator, cultivate 24 hours.
5. progressively continuing to cultivate in 500mg/L, 600mg/L, 700mg/L and 800mg/L flat board, finally obtain and be resistant to
The aimed strain of 800mg/L organophosphorus pesticides (chlopyrifos).
2nd, blended induction, domestication
1. blended dilution process
Chlopyrifos, carbendazim, procymidone and dimethomorph is dense into 6.2% with pH7.2 phosphate buffered salines respectively
The solution of degree.
2. being 6.2% four kinds of the pesticide solutions by active constituent content, respectively take 3.60mL to be well mixed, obtain mixing agriculture
Medicine.
3. blended 0.97mL, 1.30mL, 1.61mL, 1.94mL, 2.26mL, 2.58mL is taken to be separately added into 200mL pancreases
In peptone agar, it is well mixed, is configured to 300mg/L, 400mg/L, 500mg/L, 600mg/L, 700mg/L and 800mg/L
Blended tryptone agar culture medium, and flat board is made.
4. the aimed strain for being resistant to 800mg/L organophosphorus pesticides (chlopyrifos) is transferred to the flat of 300mg/L blendeds
In plate.The flat board being inoculated with is placed in 37 DEG C of constant incubators, cultivated 24 hours.
5. stepping up blended concentration in flat board, continue to induce, tame, obtain one plant of degradable high concentration mixing agriculture
The bacterial strain of medicine, is designated as FY-7, and chlorpyrifos degradation and carbendazim contrast effect are as shown in Figure 1 before and after inducing and acclimating.
It is common that above-mentioned bacterial strains FY-7 has been preserved in China Committee for Culture Collection of Microorganisms on December 30th, 2016
(abbreviation CGMCC, address are at microorganism center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number CGMCC
No.13523, Classification And Nomenclature are spindle lysine bacillus (Lysinibacillus fusiformis).
6. rinsed with sterile phosphate buffer by obtained FY-7 thalline are tamed from culture dish, with repeatedly from
The method washing thalline of heart resuspension 3 times, weighs, and is 0.1g by thalline and phosphate buffer (pH7.2):500 μ L ratio weight
New suspension thalline.50% glycerine water solution of sterilizing and bacterium solution are pressed 1:Dispensed after 1 mixing, often the μ L of pipe 500, it is cold in 4 DEG C of refrigerators
Hide.
Example 3, using spindle lysine Bacillus strain FY-7 prepare biodegradable enzyme preparation
1. strain amplification cultivation：The strain of refrigeration is taken to be inoculated into the sterile TSB culture mediums of 100mL, 37 DEG C, 180rpm trainings
Support 24h, as first order seed；First order seed is forwarded in 1L TSB culture mediums by 10% inoculum concentration, 37 DEG C, 180rpm trainings
Support 24 hours, as secondary seed.Secondary seed is forwarded in 10L industrial culture medium by 10% inoculum concentration, 37 DEG C,
180rpm cultivates 24h, as three-level seed.
2. three-level seed is inoculated into fermentation tank, inoculum concentration is the 5% of fermentation medium.It is 2.5m to control throughput3/
H, temperature be 37 DEG C, pH be 7.2 ± 0.4, rotating speed 180rpm, every when sample, determine OD600And pH, centre is according to foam situation
Sterile defoamer is added in right amount.Lower tank after fermenting 18 hours.
3. by zymotic fluid high speed centrifugation (14000rpm, temperature are 4 DEG C) 15-20min in fermentation tank, liquid is abandoned, is collected
Thalline.Thalline is pressed 1 with phosphate buffer:10 (g/mL) suspend again, obtain bacteria suspension.
4. using high pressure cell cracker, in 4-6 DEG C of 2.5-5.0MPa, temperature, the resuspension thalline of bacteria suspension is crushed, crushes two
It is secondary, obtain broken liquid.
5. broken liquid centrifuges 50min in 8000rpm (4 DEG C of temperature), supernatant is biodegradable enzyme crude enzyme liquid.
6. filtering：First with 6-8 layers filter paper filtering crude enzyme liquid, then with aperture it is 0.45 μm of membrane filtration, you can must clarify
Biodegradable enzyme solutions.
7. stabilization processes：Biodegradable enzyme solutions are diluted 100 times with phosphate buffer (pH7.2), add 0.01-
0.02% polyhexamethylene guanide, then be thoroughly mixed together in equal volume with the glycerine after sterilizing, 4 DEG C of preservations.
The component of the industrial culture medium (i.e. fermentation medium) is as follows：Fermentation medium is by tryptone described in per L
17g, phytone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 5g and water composition, volume is supplied with water.
The application of example 4, biodegradable enzyme preparation in vegetables (fruit) residues of pesticides of degrading
More representational vegetables cucumber is bought from wholesale vegetable market (Shouguang) and Lettuce is tested, first will be real
Example 3 obtains biodegradable enzyme solutions (enzyme preparation) and is added in pure water to obtain experimental liquid, and the concentration of enzyme preparation is 2.5-4ppm,
Corresponding vegetables are soaked in 3-5min in experimental liquid under 37-43 DEG C of water bath with thermostatic control, sending third party testing agency, (promise peace strength can business
(Qingdao) Co., Ltd is surveyed in product examine) detected.Cucumber and naked oats chemical residual degradation species (Boscalid, Mobucin, phonetic bacterium
Fat, dimethomorph, procymidone, metalaxyl, Propamocarb etc.) and effect is as shown in Figures 2 and 3.
Sequence table
<110>Xi'an Rutgers bio tech ltd
<120>The application of spindle lysine Bacillus strain, enzyme preparation and its degrading pesticide residues
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 947
<212> DNA
<213> Lysinibacillus fusiformis
<400> 1
ggcggctggc tccaaaaggt tacctcaccg acttcgggtg ttacaaactc tcgtggtgtg 60
acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat ccgcgattac 120
tagcgattcc ggcttcatgt aggcgagttg cagcctacaa tccgaactga gaacgacttt 180
atcggattag ctccctctcg cgagttggca accgtttgta tcgtccattg tagcacgtgt 240
gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc tccggtttgt 300
caccggcagt caccttagag tgcccaacta aatgatggca actaagatca agggttgcgc 360
tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca tgcaccacct 420
gtcaccgttg cccccgaagg ggaaactata tctctacagt ggtcaacggg atgtcaagac 480
ctggtaaggt tcttcgcgtt gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc 540
cccgtcaatt cctttgagtt tcagtcttgc gaccgtactc cccaggcgga gtgcttaatg 600
cgttagctgc agcactaagg ggcggaaacc ccctaacact tagcactcat cgtttacggc 660
gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgcgcct cagcgtcagt 720
tacagaccag aaagtcgcct tcgccactgg tgttcctcca aatctctacg catttcaccg 780
ctacacttgg aattccactt tcctcttctg cactcaagtc ccccagtttc caatgaccct 840
ccacggttga gccgtgggct ttcacatcag acttaaagga ccgcctgcgc gcgctttacg 900
cccaataatt ccggacacgc ttgccaccta cgtattaccg cggctgc 947
<210> 2
<211> 19
<212> DNA
<213>Artificial synthesized ()
<400> 2
tccgtaggtg aacctgcgg 19
<210> 3
<211> 19
<212> DNA
<213>Artificial synthesized ()
<400> 3
tcctccgctt attgatatg 19
Claims (10)
- A kind of 1. spindle lysine bacillus (Lysinibacillus fusiformis) bacterial strain, it is characterised in that：The bacterium The deposit number of strain is CGMCC No.13523.
- A kind of 2. biodegradable enzyme, it is characterised in that：The biodegradable enzyme is to utilize spindle lysine bacillus (Lysinibacillus fusiformis) bacterial strain extracts thalline endocellular enzyme and manufactured, the bacterial strain after carrying out fermented and cultured Deposit number be CGMCC No.13523.
- A kind of 3. preparation method of biodegradable enzyme, it is characterised in that：Comprise the following steps：By spindle lysine bacillus After (Lysinibacillus fusiformis) bacterial strain is fermented, thalline, broken born of the same parents, centrifugation are collected, supernatant is extracted, obtains Biodegradable enzyme solutions, the deposit number of the bacterial strain is CGMCC No.13523.
- A kind of 4. preparation method of biodegradable enzyme according to claim 3, it is characterised in that：The culture that the fermentation uses Base includes tryptone 17-19g/L, phytone 1-4g/L, sodium chloride 2-5g/L, dipotassium hydrogen phosphate 2-5g/L and glucose 2-5g/L, Medium's PH Value 6.8-7.6.
- A kind of 5. preparation method of biodegradable enzyme according to claim 3, it is characterised in that：The condition of the fermentation be in 34-39 DEG C, 180-200rpm culture 16-24 hours.
- A kind of 6. preparation method of biodegradable enzyme according to claim 3, it is characterised in that：After the fermentation, it will send out Ferment product centrifuges 15-25min in 12000-14000rpm, collects thalline, height crushes born of the same parents after thalline is resuspended, then in 8000- 10000rpm centrifuges 50-65min, collects supernatant, by supernatant liquid filtering, obtains biodegradable enzyme solutions.
- A kind of 7. preparation method of biodegradable enzyme according to claim 6, it is characterised in that：The specific steps of the filtering For by supernatant, successively through filter paper and 0.45 μm of membrane filtration, it is biodegradable enzyme solutions to obtain supernatant liquid.
- A kind of 8. biodegradable enzyme preparation, it is characterised in that：The active component of the enzyme preparation is spindle lysine bacillus (Lysinibacillus fusiformis) bacterial strain or the biodegradable enzyme by the bacterial strain biosynthesis, the preservation of the bacterial strain Numbering is CGMCC No.13523.
- A kind of 9. spindle lysine bacillus (Lysinibacillus fusiformis) bacterium as claimed in claim 1 Application of the strain in degrading pesticide residues.
- A kind of 10. application of the biodegradable enzyme preparation in degrading pesticide residues as claimed in claim 8.
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Cited By (2)
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| CN109504622A (en) * | 2018-11-16 | 2019-03-22 | 广东植物龙生物技术股份有限公司 | A kind of lysine bacillus and the composite bacteria agent of bacillus subtilis and preparation method thereof |
| CN109576171A (en) * | 2018-11-16 | 2019-04-05 | 广东植物龙生物技术股份有限公司 | Spindle lysine bacillus and its application |
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| CN102134559A (en) * | 2010-11-15 | 2011-07-27 | 广州吉家庄生物科技有限公司 | Bacillus brevis, method for extracting bacillus brevis esterase and application of same |
| CN106566793A (en) * | 2016-11-14 | 2017-04-19 | 成都医学院 | Lysinibacillus sp. and application thereof in pesticide degrading |
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- 2017-08-29 CN CN201710757505.6A patent/CN107384834B/en not_active Expired - Fee Related
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| CN102134559A (en) * | 2010-11-15 | 2011-07-27 | 广州吉家庄生物科技有限公司 | Bacillus brevis, method for extracting bacillus brevis esterase and application of same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109576171A (en) * | 2018-11-16 | 2019-04-05 | 广东植物龙生物技术股份有限公司 | Spindle lysine bacillus and its application |
| CN109504622B (en) * | 2018-11-16 | 2022-01-07 | 广东植物龙生物技术股份有限公司 | Composite microbial inoculum of lysine bacillus and bacillus subtilis and preparation method thereof |
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|---|---|
| CN107384834B (en) | 2020-05-22 |
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