CN116875511A - Pseudomonas taiwan Pt16 strain and application thereof - Google Patents
Pseudomonas taiwan Pt16 strain and application thereof Download PDFInfo
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- CN116875511A CN116875511A CN202310990783.1A CN202310990783A CN116875511A CN 116875511 A CN116875511 A CN 116875511A CN 202310990783 A CN202310990783 A CN 202310990783A CN 116875511 A CN116875511 A CN 116875511A
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- 241000589516 Pseudomonas Species 0.000 title abstract description 26
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 claims abstract description 103
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 claims abstract description 103
- 229960002715 nicotine Drugs 0.000 claims abstract description 103
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 77
- 241000208125 Nicotiana Species 0.000 claims abstract description 66
- 241001468880 Pseudomonas taiwanensis Species 0.000 claims abstract description 61
- 244000061176 Nicotiana tabacum Species 0.000 claims abstract description 11
- 244000005700 microbiome Species 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 38
- 230000001580 bacterial effect Effects 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 15
- 230000000593 degrading effect Effects 0.000 claims description 14
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 229910000859 α-Fe Inorganic materials 0.000 claims description 5
- 230000006872 improvement Effects 0.000 claims description 4
- 238000009395 breeding Methods 0.000 claims description 3
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- 238000002474 experimental method Methods 0.000 description 6
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
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- 238000004659 sterilization and disinfection Methods 0.000 description 3
- MYKUKUCHPMASKF-VIFPVBQESA-N (S)-nornicotine Chemical compound C1CCN[C@@H]1C1=CC=CN=C1 MYKUKUCHPMASKF-VIFPVBQESA-N 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 206010013911 Dysgeusia Diseases 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 2
- MYKUKUCHPMASKF-UHFFFAOYSA-N Nornicotine Natural products C1CCNC1C1=CC=CN=C1 MYKUKUCHPMASKF-UHFFFAOYSA-N 0.000 description 2
- 241000589776 Pseudomonas putida Species 0.000 description 2
- 239000000589 Siderophore Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000208292 Solanaceae Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
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- 229910052742 iron Inorganic materials 0.000 description 2
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- 238000005507 spraying Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000394642 Pseudomonas marginalis pv. marginalis Species 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
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- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
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- 235000006408 oxalic acid Nutrition 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
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- 239000012138 yeast extract Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses a Pseudomonas taiwanensis Pt16 strain and application thereof, which is obtained by screening soil of a tobacco planting place in Yongzhou of Hunan, and is named as Pseudomonas taiwanensis Pt16 after separation and identification. It was deposited at 20/6/2022 with the microorganism collection center of Guangdong province under the accession number GDMCC No:62554.CAS flat plate detection results show that the bacterium has obvious ferriphilic ability and is a strain of ferriphilic bacteria; meanwhile, the tobacco smoke-cured tobacco nicotine can be degraded, and the quality of the tobacco smoke-cured tobacco is improved. The culture medium is from a soil environment, the raw materials of the culture medium are simple and easy to obtain, the preparation process of the culture medium is simple, the energy consumption is low, and the cost is low; the sprayed culture solution has higher environmental safety, little harm to the environment, almost no secondary pollution, almost no change of soil matrix and no influence on the soil structure. The Pseudomonas taiwan Pt16 is used in tobacco planting soil, tobacco plants and tobacco products, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of nicotine degradation, in particular to a Pseudomonas taiwan Pt16 strain and application thereof.
Background
Tobacco (Nicotiana tabacum L.) is an important economic crop in China, tobacco is an alkaloid-containing crop, and the alkaloids mainly comprise 4 kinds of nicotine, nornicotine, neonicotinoid and pseudoscouring rush alkaloid, wherein the nicotine accounts for more than 94% of the total alkaloid content of the tobacco.
Nicotine (also known as Nicotine, formula C) 10 H 14 N 2 Is a toxic heterocyclic compound with stable structure and composed of pyridine ring and pyrrole ring, and widely exists in various Solanaceae (Solanaceae) plants, and is an important factor for evaluating tobacco and cigarette quality. Nicotine is a key component of smoking addiction and also has some degree of damage to the human body. With the mass production and consumption of tobacco products, tobacco waste, including nicotine, is introduced into the environment, which, if improperly treated, can be detrimental to human health and the environment.
In recent years, as the yield is pursued on one side, the high content of nicotine in flue-cured tobacco leaves in partial production areas in China is too high due to the fact that a large amount of nitrogenous fertilizer is applied, especially the nicotine content of upper tobacco leaves is too high to be used in cigarette formulas, and the upper tobacco leaves in many cigarette factories are seriously backlogged. The nicotine content of the burley tobacco in China is much higher than that in the United states, and the development of the domestic mixed cigarettes is affected by the nicotine content. Therefore, reducing the nicotine content of tobacco leaves is one of the problems to be solved urgently in the tobacco industry.
At present, physical and chemical treatment methods are mainly concentrated on the degradation of nicotine. The microbial degradation bacteria have the characteristics of strong degradation capability, rich functional microorganism types, various degradation paths, small influence on environment and the like, and are a low-cost and high-efficiency nicotine treatment method. The microorganism nicotine-degrading bacteria belong to the genera Pseudomonas (Pseudomonas), arthrobacter (Acetobacter), pallidum (Palebsiella), agrobacterium (Agrobacterium).
The reduction of nicotine content in tobacco leaves by microbial degradation has been studied and experienced for many years at home and abroad.
The Brown and Williamson tobacco company utilizes pseudomonas to degrade nicotine in tobacco, and found that after the mixed tobacco shreds (1:1) of burley tobacco and flue-cured tobacco are treated by pseudomonas liquid for 18 hours, the nicotine content is reduced to 0.85 percent from 2.00 percent on average, and the nicotine content of each cigarette is reduced to 0.98mg from 1.58 mg; microorganisms isolated from the surface of tobacco seeds can degrade nicotine into formamide, ammonia, oxalic acid, and trace amounts of malonic acid and succinic acid; separating type A and type B bacteria from soil, wherein the type A bacteria only can degrade nicotine, the type B bacteria not only can degrade nicotine, but also can degrade nornicotine and pseudoscouring, and the type B bacteria belong to pseudomonads (G-); isolating from a cigar leaf a pseudomonad malodor (Pseudomonas putida, G-) that degrades nicotine and a cellomonas sp that removes both nicotine and nitrate; separating a highly-efficiently degraded bacterial strain Agrobacter sp.strain S33 from tobacco rhizosphere soil, wherein 1g/L nicotine can be completely degraded within 6 hours under optimal culture conditions; strains Pseudomonas marginalis ND are obtained from tobacco soil in Hunan province, and nicotine in 1g/L liquid culture medium can be degraded by 70.40% in 2 d; 3 strains with strong degradation capability to nicotine are separated from tobacco leaves.
The method for degrading the nicotine by microorganisms is low in cost, high in efficiency and free of secondary pollution, so that the technology for degrading the nicotine has important significance in the aspects of reducing the content of the nicotine, reducing the pollution of the nicotine, improving the utilization rate of tobacco waste, protecting water resources, protecting ecological environment and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a Pseudomonas taiwan Pt16 strain and application thereof. The strain is obtained from tobacco land through screening, purifying, identifying and other technologies, and through a nicotine culture medium and a tobacco strain spraying test, and the nicotine degradation efficiency is measured, so that the strain is a beneficial strain with high-efficiency nicotine degradation, can degrade nicotine efficiently and improve baking, and has good practical application value.
Quality of smoke
The first object of the invention is to provide a strain of Pseudomonas taiwanensis (Pseudomonas taiwanensi s) Pt16.
The second object of the invention is to provide the application of the Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain and/or the bacterial liquid thereof in degrading nicotine or preparing a nicotine degradation product.
A third object of the invention is to provide the use of the pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain and/or a bacterial liquid thereof for reducing the nicotine content of tobacco.
The fourth object of the invention is to provide the application of the Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain and/or bacterial liquid thereof in flue-cured tobacco quality improvement breeding.
The fifth object of the invention is to provide the application of the Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain and/or the bacterial liquid thereof in the production of ferrites.
It is a sixth object of the present invention to provide a formulation for degrading nicotine.
It is a seventh object of the present invention to provide a method of degrading nicotine or reducing the nicotine content of tobacco.
In order to achieve the above object, the present invention is realized by the following technical scheme:
the invention claims a Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain which is deposited in the Guangdong province microorganism strain collection at 2022, 6 and 20 days, and has a strain deposit number of GDMCC NO:62554 the strain can degrade nicotine with high efficiency and improve quality of flue-cured tobacco
The following are further claimed:
the application of the Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain and/or bacterial liquid thereof in degrading nicotine or preparing a nicotine degradation product.
The application of the Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain and/or the bacterial liquid thereof in reducing the nicotine content of tobacco.
The application of the Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain and/or bacterial liquid thereof in flue-cured tobacco quality improvement breeding.
The Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain and/or the bacterial liquid thereof are applied to the production of the ferrites.
Also claimed is a formulation for degrading nicotine comprising said Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain and/or a bacterial liquid thereof.
A method for degrading nicotine or reducing the nicotine content of tobacco, which adopts the pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain and/or bacterial liquid thereof to treat a sample.
Preferably, the bacterial liquid is fermentation liquid with the concentration of 1 multiplied by 10 8 ～1×10 10 cfu/mL。
Preferably, the tobacco plants are sprayed with the fermentation broth.
Preferably, the treatment is performed during the topping period of the tobacco.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a pseudomonad taiwan for degrading nicotine and application thereof, which are obtained by screening soil of tobacco planting places in Yongzhou of Hunan province, and are named as pseudomonad taiwan (Pseudomonas taiwanensis) Pt16 after separation and identification. It was deposited at 20/6/2022 with the microorganism collection center of Guangdong province under the accession number GDMCC No:62554.CAS flat plate detection results show that the bacterium has obvious ferriphilic ability and is a strain of ferriphilic bacteria; meanwhile, the tobacco smoke-cured tobacco nicotine can be degraded, and the quality of the tobacco smoke-cured tobacco is improved.
The strain comes from soil environment, the culture medium raw materials are simple and easy to obtain, the preparation process of the culture solution is simple, the energy consumption is low, and the cost is low; the sprayed culture solution has higher environmental safety, little harm to the environment, almost no secondary pollution, almost no change of soil matrix and no influence on the soil structure. The Pseudomonas taiwan Pt16 is used in tobacco planting soil, tobacco plants and tobacco products, and has good application prospect.
Drawings
FIG. 1 is a photograph of a Pt16 isolate plate.
FIG. 2 is a photograph of a Pt16 LB plate; a is the original colony size; b is the size of the colony of 7.5.
FIG. 3 is a Pt16 phylogenetic analysis; and (3) injection: marked on each branch is: genBank serial number + strain name.
Fig. 4 is a graph of Pt16 degradation of nicotine in a nicotine medium.
FIG. 5 shows the degradation of nicotine in LB medium by Pt16.
FIG. 6 shows the degradation of nicotine on Pt16 strain tobacco plants.
FIG. 7 shows the Pt16 siderophore assay.
Detailed Description
The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 isolation culture of Strain Pt16
1. Preparation of Selective separation Medium
LB medium (10 g peptone, 5g yeast extract, 10g NaCl,15g agar, dissolved in 1L sterile ddH) 2 Adjusting pH to 7.0 in O water, and autoclaving at 121deg.C for 20 min);
preparing a microelement solution: 0.4g MnSO 4 ·7H 2 O，0.2g CaCl 2 ·2H 2 O，0.2g FeSO 4 ·7H 2 O was dissolved with 0.1mol/L HCl and the volume was set to 100mL.
Selective medium (2 g/L): 13.3g K 2 HPO 4 ，4g KH 2 PO 4 ，0.2g MgSO 4 ·7H 2 O,0.5mL of trace element solution, 18g of agar, pH was adjusted to 7.0, and autoclaved at 121℃for 20 minutes. After sterilization, 2.2mL of liquid nicotine with purity of 90% filtered by a 0.22 μm filter membrane is added when the temperature is about 70 ℃.
Selective medium (3 g/L): 13.3g K 2 HPO 4 ，4g KH 2 PO 4 ，0.2g MgSO 4 ·7H 2 O,0.5mL trace element solution, 18g agar, pH was adjusted to 7.0, 121 DEG CAutoclaving for 20 minutes. After sterilization, 3.3mL of liquid nicotine with purity of 90% filtered by a 0.22 μm filter membrane is added when the temperature is about 70 ℃.
2. Screening nicotine degrading bacteria from soil
The selective medium for separation uses nicotine as the only carbon source and nitrogen source, the concentration of nicotine is 2g/L and 3g/L, and the strain growing on the selective medium has the capability of utilizing nicotine.
The specific process is as follows: 1.0g of soil sample is weighed and added into a triangular flask filled with 30mL of PBS, and enrichment culture is carried out for 48 hours at the temperature of 30 ℃ and the rotating speed of 150r/min by a shaking table. 1mL of the supernatant is sucked in an ultra-clean bench, added into a conical flask filled with 30mL of selective medium, subjected to domestication culture for 48 hours at the temperature of 30 ℃ by a shaking table and the rotating speed of 150r/min, and then separated by a dilution plate on a nicotine solid medium. Sucking 1mL of supernatant to obtain stock solution, adding into a test tube containing 9mL of sterilized PBS, and shaking to obtain 10 -1 A multiplied bacterial liquid; preparation of 10 by the same method -2 、10 -3 、10 -4 、10 -5 、10 -6 The bacterial liquid of multiple times is taken to be 0.1mL 10 -3 、10 -4 、10 -5 、10 -6 The multiple bacteria solution was diluted 3 times, and each glass bead was spread on a plate of the prepared selective medium, and cultured at 30℃for 4 days. Individual colonies were selected for further streaking and the procedure repeated until pure single strains were obtained and numbered as shown in figure 1.
3. Continuous purification culture and photographing
After single colonies grow out in the selective medium, each single colony is continuously streaked and purified for at least more than 5 times on an LB plate, then the streaked plate of the isolated strain is photographed (wherein a photographing diagram of the strain with the number of Pt16 is shown in figure 2) and transferred into an LB liquid medium, and the single colony is stored in a 15% glycerol aqueous solution when being shaken to an exponential growth phase and is frozen in a refrigerator at the temperature of minus 80 ℃ for standby.
Example 2 identification of Strain Pt16 and phylogenetic analysis
1. Colony morphology characterization
From the morphological point of view, the Pt16 strain is shown in FIG. 2B, and forms a round opaque gray-white colony with neat edges on an LB plate, and is a gram-negative bacterium, and the optimal growth temperature is 30 ℃; the nutrition requirement is not high, and the growth is good on the common culture medium and the nicotine culture medium.
2. Molecular biological identification
1. Experimental method
(1) The genomic DNA of the preserved Pt16 strain was extracted using a bacterial genomic DNA extraction kit (TIANamp Bacteria DNAKit) from Tiangen biosystems, and 16S rDNA was amplified using the extracted DNA as a template and 16S rDNA universal primers 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers, and the PCR reaction system was as shown in Table 1.
TABLE 1 bacterial 16S rDNA PCR amplification System (20. Mu.L)
After gentle mixing, the mixture was centrifuged briefly and placed on a PCR instrument according to: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 15s, extension at 72℃for 15s,35 cycles; 72 ℃ for 5min; the procedure of end reaction at 4℃was followed by 1.5% agarose gel detection and gel cutting recovery purification of the PCR product, which was then sequenced by the engine biotechnology Co., ltd.
(2) Strain Pt16 phylogenetic analysis
The amplified product of strain Pt16 was sequenced using bacterial universal primers 27F and 1492R, and after the sequencing results were returned, the sequencing quality was analyzed using SeqMan software and the forward and reverse sequences were spliced. After splicing, performing Blast comparison on the obtained 16SrDNA gene sequence at NCBI, selecting a strain sequence with a relatively close relationship, constructing a phylogenetic tree by using a software MEGA 7 through a neighbor-Joing method, adjusting bootstrap values, and checking the reliability of the evolutionary tree.
2. Experimental results
The nucleotide sequence of the 16S rDNA gene is shown as SEQ ID NO. 1, the development analysis result is shown as figure 3, and the similarity between Pt16 and Pseudomonas putida strain NBRC is the highest as can be seen from figure 3. It was designated as a pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain which was deposited at the cantonese province microbiological bacterial collection center (GDMCC) at 20/6/2022 under the accession number GDMCCNo:62554, classified and named Pseudomonas taiwanensis Pt, and the preservation address is 100 # of Xian Liang Zhonglu in Guangzhou City of Guangdong province.
EXAMPLE 3 degradation of nicotine in a nicotine Medium by Pseudomonas taiwan Pt16 Strain
1. Experimental method
(1) And (3) nicotine standard curve preparation: seven concentration gradient standard solutions of 250mg/mL, 500mg/mL, 750mg/mL, 1000mg/mL, 1250mg/mL and 1500mg/mL are prepared from a 99.8% purity nicotine standard substance, and a nicotine concentration standard curve is established by high performance liquid chromatography.
(2) 200mL of a selective medium with a nicotine content of 2g/L was prepared, and an experimental group was set as a medium for inoculating Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain (strain deposit number is GDMCC No: 62554) and a control group was a nicotine liquid medium without inoculating the strain. The experimental group and the control group were cultured in an incubator at 30 ℃.
(3) The detection method comprises the following steps: culturing the experimental group and the control group in a 30 ℃ incubator, sampling at 0h, 5h, 10h, 15h, 20h, 25h and 30h respectively, and taking 2mL of bacterial liquid culture medium in an ultra-clean bench respectively, wherein 1mL of bacterial liquid culture medium is filtered out by a filter membrane with the pore diameter of 0.22 mu m, so as to obtain the liquid to be detected.
The nicotine content detection adopts high performance liquid chromatography, and the chromatographic column is: agilentTC-C18; mobile phase: methanol to 0.02mol disodium hydrogen phosphate buffer (ph=4.2) in a volume ratio of 10:90; the column temperature is 30 ℃, the flow rate is 1mL/min, the sample injection amount is 10 mu L, and the detection wavelength is 259nm. 1mL of the cells were centrifuged at 1000g for 2min and resuspended in PBS. OD determination with an ELISA apparatus 600 The microplate reader was set at 200 μl per well and 3 replicates were set.
2. Experimental results
The nicotine content was measured at 0h, 5h, 10h, 15h, 20h, 25h, 30h by HPLC. The test results are shown in FIG. 4, and the test shows that the nicotine content in the culture medium of the test group starts to decrease within 0h and decreases by 99% within 15h, thus proving that the Pseudomonas taiwan (Pseudomonas taiwanensis) Pt16 strain (with the strain deposit number of GDMCC No. 62554) can utilize the ability of using nicotine as the sole carbon and nitrogen source for growth.
EXAMPLE 4 degradation of nicotine in LB Medium by Pseudomonas taiwan Pt16 Strain
1. Experimental method
(1) And (3) standard curve preparation: seven concentration gradient standard solutions of 250mg/mL, 500mg/mL, 750mg/mL, 1000mg/mL, 1250mg/mL and 1500mg/mL are prepared from a 99.8% purity nicotine standard substance, and a nicotine concentration standard curve is established by high performance liquid chromatography.
(2) The culture method comprises the following steps: 200mL of LB medium was prepared and 90% pure nicotine was added to make it a LB medium containing 2g/L of nicotine. An experimental group was set up in which an LB medium containing 2g/L of nicotine was inoculated with Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (accession No. GDMCC No: 62554), and a control group in which an LB medium containing 2g/L of nicotine was not inoculated. The experimental group and the control group were cultured in an incubator at 30 ℃.
(3) Bacterial liquid thallus and nicotine content: in an ultra-clean bench, respectively taking bacterial solutions from an experimental group and a control group at 0h, 2.5h, 5h, 7.5h and 10h,
and (3) taking 1mL of bacterial liquid, and filtering out bacteria in the bacterial liquid by using a filter membrane with the aperture of 0.22 mu m to obtain a liquid to be detected, wherein the liquid to be detected is used for detecting the nicotine content.
The nicotine content detection adopts high performance liquid chromatography, and the chromatographic column is: agilentTC-C18; mobile phase: methanol to 0.02mol disodium hydrogen phosphate buffer (ph=4.2) in a volume ratio of 10:90; the column temperature is 30 ℃, the flow rate is 1mL/min, the sample injection amount is 10 mu L, and the detection wavelength is 259nm.
1mL of the cells were further collected, centrifuged at 1000g for 2min, and resuspended in PBS. OD determination with an ELISA apparatus 600 The microplate reader was 200 μl and 3 replicates per well.
2. Experimental results
The degradation result of nicotine in LB medium is shown in figure 5, and the nicotine content in the culture medium of the experimental group is found to be reduced by more than 50% within 5h and more than 95% within 10h through detection. The Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (with the strain deposit number of GDMCC No. 62554) still can preferentially utilize nicotine as a metabolic substance under the condition of containing other carbon and nitrogen sources, so that the strain has strong nicotine metabolism capability.
EXAMPLE 5 degradation of Nicotine on tobacco plants by Pseudomonas taiwan Pt16 Strain
1. Experimental method
(1) Culture and arrangement of bacterial cells
1L of Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (accession number GDMCC No: 62554) was prepared as a bacterial liquid (1×10) 8 ～1×10 10 cfu/mL) for spraying.
(2) Treatment of tobacco plants with Pseudomonas taiwan Pt16 strain
One mu of tobacco (cloud tobacco) was planted until the topping period (as shown in fig. 6A). After topping the tobacco, 1L of bacterial liquid is diluted into 15L of water to spray the leaves in the middle of the tobacco, and the tobacco is used as an experimental group. The control group was sprayed with an equal amount of clear water as a control. After bacterial liquid (or clear water) is sprayed, randomly taking the fourth leaf of the tobacco plant at 0h, 24h, 30h, 48h and 120h respectively, and detecting the nicotine content.
(3) Detection of nicotine content
Immediately weighing fresh tobacco leaves after taking off, drying (105 ℃,30min;70 ℃ for 6 hours), and weighing dry weight after drying; grinding the dried tobacco leaves, and sieving the tobacco leaves with a 100-mesh sieve to obtain tobacco powder; 0.1g of tobacco powder and 0.2g of activated carbon are taken into a 150mL conical flask, and 25mL of 0.5mol/L hydrochloric acid is added into the conical flask; heating the alcohol lamp to boil, and keeping boiling for 5min; after the solution is cooled, a 250mL volumetric flask is used for constant volume; after constant volume, filtering, and discarding the first 10ml of filtrate; and (3) carrying out color comparison on 30mL of filtrate at 236nm, 259nm and 282nm by using a spectrophotometer, measuring the light absorption value of the filtrate, and then calculating to obtain the nicotine content according to the following formula: nicotine (mg/g) =1.059× [ A ] 259 -0.5×(A 236 +A 282 )]XV.times.1000/[ number of samples X (1-moisture content). Times.34.3.times.1000)]。
2. Experimental results
The result is obtained from the detection and calculation as shown in fig. 6B. The experimental group had a decrease in nicotine content of about 53% at 24h compared to the control group, and by 120h the nicotine content was still about 38% lower compared to the control group, although the nicotine content was gradually supplemented back. The results show that Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (accession number GDMCC No. 62554) also significantly degrades nicotine on tobacco plants.
EXAMPLE 6 sensory evaluation of tobacco treated with Pt16 Strain
1. Experimental method
The Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (with the strain deposit number of GDMCCNo: 62554) is used for degrading nicotine on the cured tobacco of the cloud 87 of the Yongzhou production area of 2022.
The specific method comprises the following steps: bacterial solution of Pseudomonas taiwanensis Pt16 strain (with the strain deposit number of GDMCC No. 62554) is added at a concentration of 1×10 8 cfu/mL is uniformly sprayed on the redried tobacco flakes, the tobacco leaves are continuously and gently turned over, and balanced moisture is kept stand for 30 minutes; after balancing the water, the tobacco leaves are placed in indoor shade and shading treatment is carried out for 3 days. After the treatment is finished, tobacco leaves are shredded and dried, the moisture of the tobacco leaves is controlled to be 12% -12.5%, sensory quality evaluation is carried out, and the same batch of samples which are not treated are used as a control.
Wherein, the sensory quality score= (aroma quality x 0.35+aroma amount x 0.25+miscellaneous gas x 0.1+pungent x 0.15+aftertaste x 0.15) ×11.11.
Fragrance quality: good, better (7.6-9.0), middle-upper (6.1-7.5), middle (4.6-6.0), middle-lower (3.1-4.5), worse (less than or equal to 3);
fragrance amount: feet (7.6-9.0), feet (6.1-7.5), feet (4.6-6.0), less feet (3.1-4.5), less feet (less than or equal to 3);
miscellaneous gas: very light (7.6-9.0), lighter (6.1-7.5), light (4.6-6.0), heavier (3.1-4.5) and heavy (less than or equal to 3);
irritation: small (7.6-9.0), small (6.1-7.5), large (3.1-4.5) and large (less than or equal to 3);
aftertaste: comfortable (7.6-9.0), more comfortable (6.1-7.5), shang Shi (4.6-6.0), underfit (3.1-4.5) and tongue stagnation (less than or equal to 3).
2. Experimental results
The results are shown in Table 2, after Pt16 strain treatment, the cloud tobacco 87 style characteristic index of the producing area in Yongzhou of 2022 can be obviously improved, the smoke concentration and strength are properly reduced, the smoke comfort is improved, and the formula applicability of tobacco leaves is improved. The treatment of Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (with the strain deposit number of GDMCC No. 62554) has a remarkably improved quality characteristic index on cloud 87 of the producing area of 2022.
TABLE 2 degradation of nicotine by Pt16 Strain on Yongzhou Yunyan 87 redried strips
Example 7 test of the ferrite-producing ability of Pseudomonas taiwan Pt16 Strain
1. Experimental method
Strains were tested for their capacity to produce iron carriers using CAS plates. The CAS panel configuration method is as follows: the modified CAS agar kit PM0821 (Coolaber) was purchased, 141g was weighed, deionized water was added to a volume of 800mL, and the mixture was dissolved by magnetic stirring. Sterilizing with high pressure steam at 115 deg.C for 20 min. After sterilization, the medium was cooled to 60 ℃,100 ml of 10 Xbuffer and 100ml of 10 XCAS detection solution preheated at 60℃were slowly added to the medium, and the mixture was poured onto a plate without generating bubbles. Sealing with sealing film after gel, and inverting at 4deg.C for use.
The single colony is picked up after the glycerol bacteria of the Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (with the strain deposit number of GDMCC No: 62554) stored in a refrigerator at the temperature of minus 80 ℃ are streaked and inoculated into an LB liquid culture medium, the culture medium is shaken at the temperature of 30 ℃ and at 180rpm for 12 hours, four small holes are respectively punched at the four corners of a CAS flat plate by a 1cm puncher, and 2mL of bacterial liquid is dripped into the small holes. The CAS plates inoculated with only the empty LB liquid medium were used as controls, and were placed in a constant temperature incubator at 30℃for 72 hours, and the color change around the colonies was observed.
2. Experimental results
The results of the test for the ferrite-producing ability of Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (accession number GDMCCNo: 62554) are shown in FIG. 7.
As can be seen from FIG. 7, after 72h of incubation at 30℃the Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (GDMCC No: 62554) had a pronounced yellow halo at the inoculation of the inoculum solution on the CAS plate, indicating that the Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (accession number GDMCCNo: 62554) can produce a siderophore with high affinity for iron ions, which are secreted by the Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (accession number GDMCC No: 62554) in the CAS plate, thus exhibiting a yellow halo. Since the fermentation broth of Pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain (with the strain deposit number of GDMCC No. 62554) contains ferriphile, the fermentation broth is used for preventing and treating plant diseases.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
SEQ ID NO:1～1423bp
ATGCTACCTGCAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGG
GTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCT
AATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGA
TGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCG
TAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTA
CGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCC
GCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGT
AAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGC
CAGCAGCCGCGGTAATCAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG
CGCGTAGGTGGTTCGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCA
TCCAAAACTGGCGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTG
AAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACT
GACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCAC
GCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACG
CATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACG
GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTT
ACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCT
GACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC
GTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGA
GACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTT
ACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGA
GGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTG
CGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCG
GGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGT
CTAACCTTCGGGAGGACGGTACCACGGTGATCAG
Claims (10)
1. A strain of pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 deposited at the microorganism strain collection, cantonese province, at 20/6/2022 under the accession number GDMCC NO:62554.
2. use of a pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain and/or a bacterial liquid thereof according to claim 1 for degrading nicotine or for the preparation of a nicotine-degrading product.
3. Use of a pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain and/or a bacterial liquid thereof according to claim 1 for reducing the nicotine content of tobacco.
4. Use of a pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain and/or a bacterial liquid thereof according to claim 1 in flue-cured tobacco quality improvement breeding.
5. Use of a pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain and/or a bacterial liquid thereof as claimed in claim 1 for the production of ferrites.
6. A formulation for degrading nicotine, comprising the pseudomonas taiwanensis Pt16 strain and/or a bacterial liquid thereof according to claim 1.
7. A method of degrading nicotine or reducing the nicotine content of tobacco, characterized in that a sample is treated with a pseudomonas taiwanensis (Pseudomonas taiwanensis) Pt16 strain and/or a bacterial liquid thereof according to claim 1.
8. The method according to claim 7, wherein the bacterial liquid is a fermentation liquid with a concentration of 1X 10 8 ～1×10 10 cfu/mL。
9. The method of claim 8, wherein the tobacco plants are sprayed with the fermentation broth.
10. The method of claim 8, wherein the treatment is performed during a tobacco topping period.
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