CN116445335A - Arthrobacter ureafaciens Pu17 capable of efficiently degrading nicotine and application thereof - Google Patents
Arthrobacter ureafaciens Pu17 capable of efficiently degrading nicotine and application thereof Download PDFInfo
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- CN116445335A CN116445335A CN202310302513.7A CN202310302513A CN116445335A CN 116445335 A CN116445335 A CN 116445335A CN 202310302513 A CN202310302513 A CN 202310302513A CN 116445335 A CN116445335 A CN 116445335A
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- nicotine
- tobacco
- arthrobacter
- ureafaciens
- paenarthrobacter
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- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 title claims abstract description 149
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 title claims abstract description 149
- 229960002715 nicotine Drugs 0.000 title claims abstract description 149
- 241001524178 Paenarthrobacter ureafaciens Species 0.000 title claims abstract description 51
- 230000000593 degrading effect Effects 0.000 title claims abstract description 30
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 110
- 241000208125 Nicotiana Species 0.000 claims abstract description 100
- 241000186063 Arthrobacter Species 0.000 claims abstract description 54
- 230000015556 catabolic process Effects 0.000 claims description 21
- 238000006731 degradation reaction Methods 0.000 claims description 21
- 239000003124 biologic agent Substances 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 20
- 239000006152 selective media Substances 0.000 abstract description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 13
- 230000002829 reductive effect Effects 0.000 abstract description 11
- 244000061176 Nicotiana tabacum Species 0.000 abstract description 10
- 239000001963 growth medium Substances 0.000 abstract description 10
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 10
- 229910052799 carbon Inorganic materials 0.000 abstract description 7
- 239000000779 smoke Substances 0.000 abstract description 6
- 238000005507 spraying Methods 0.000 abstract description 5
- 230000004060 metabolic process Effects 0.000 abstract description 2
- 239000002207 metabolite Substances 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 23
- 230000001580 bacterial effect Effects 0.000 description 21
- 241000894006 Bacteria Species 0.000 description 19
- 239000002609 medium Substances 0.000 description 16
- 230000000813 microbial effect Effects 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 239000003513 alkali Substances 0.000 description 7
- 235000019504 cigarettes Nutrition 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 229930013930 alkaloid Natural products 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000002689 soil Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 150000003797 alkaloid derivatives Chemical class 0.000 description 4
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- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-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
- 235000019506 cigar Nutrition 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
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- 238000012360 testing method 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
- 241000194103 Bacillus pumilus Species 0.000 description 2
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 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
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- MYKUKUCHPMASKF-UHFFFAOYSA-N Nornicotine Natural products C1CCNC1C1=CC=CN=C1 MYKUKUCHPMASKF-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241000589776 Pseudomonas putida Species 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
- NMLQNVRHVSWEGS-UHFFFAOYSA-N [Cl].[K] Chemical compound [Cl].[K] NMLQNVRHVSWEGS-UHFFFAOYSA-N 0.000 description 2
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- 231100000614 poison Toxicity 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000013441 quality evaluation Methods 0.000 description 2
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- 238000012163 sequencing technique Methods 0.000 description 2
- 230000000391 smoking effect Effects 0.000 description 2
- 239000011550 stock 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
- 239000000725 suspension Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- RUFPHBVGCFYCNW-UHFFFAOYSA-N 1-naphthylamine Chemical compound C1=CC=C2C(N)=CC=CC2=C1 RUFPHBVGCFYCNW-UHFFFAOYSA-N 0.000 description 1
- AQCRXZYYMOXFAN-UHFFFAOYSA-N 2-(1-methyl-2-pyrrolidinyl)-pyridine Chemical compound CN1CCCC1C1=CC=CC=N1 AQCRXZYYMOXFAN-UHFFFAOYSA-N 0.000 description 1
- TWBPWBPGNQWFSJ-UHFFFAOYSA-N 2-phenylaniline Chemical group NC1=CC=CC=C1C1=CC=CC=C1 TWBPWBPGNQWFSJ-UHFFFAOYSA-N 0.000 description 1
- IRJNJBIOUYJBHG-UHFFFAOYSA-N 3-(1-methylpyrrolidin-2-yl)pyridine Chemical compound CN1CCCC1C1=CC=CN=C1.CN1CCCC1C1=CC=CN=C1 IRJNJBIOUYJBHG-UHFFFAOYSA-N 0.000 description 1
- 241000589220 Acetobacter Species 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- TXVHTIQJNYSSKO-UHFFFAOYSA-N BeP Natural products C1=CC=C2C3=CC=CC=C3C3=CC=CC4=CC=C1C2=C34 TXVHTIQJNYSSKO-UHFFFAOYSA-N 0.000 description 1
- 241000186321 Cellulomonas Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- 206010013911 Dysgeusia Diseases 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241000192041 Micrococcus Species 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
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 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
- 206010042618 Surgical procedure repeated Diseases 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
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- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 239000000618 nitrogen fertilizer Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
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- 239000001384 succinic acid Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- 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
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/45—Tobacco
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
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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
- C12N1/20—Bacteria; Culture media therefor
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- 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
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
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Abstract
The invention discloses Arthrobacter ureafaciens Pu17 capable of efficiently degrading nicotine and application thereof. Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 had the ability to grow on nicotine as the sole carbon and nitrogen source, and was cultured on selective medium containing 2g/L nicotine for 30h with a 80% decrease in nicotine content. Under the condition of other carbon and nitrogen sources, nicotine is preferentially used as a metabolite, the nicotine has stronger nicotine metabolism capability, and the nicotine can be completely degraded when the nicotine is cultured for 7.5 hours on LB culture medium containing 2g/L of nicotine, the nicotine content is reduced by more than 90 percent and the nicotine is 10 hours. Spraying the strain on tobacco plants for 24 hours, wherein the nicotine content is reduced by about 30 percent; the strain is sprayed on the cured tobacco sheets, so that the smoke concentration and strength of tobacco leaves are reduced, and the comfort of the smoke and the formula applicability of the tobacco leaves are improved.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to Arthrobacter urealyticum Pu17 capable of efficiently degrading nicotine 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 (Nicotine) is also known as NigulineButyl, molecular formula C 10 H 14 N 2 The compound is a toxic heterocyclic compound with stable structure and composed of pyridine ring and pyrrole ring, is widely applied to various Solanaceae (Solanaceae) plants, and is an important quality evaluation factor of tobacco and cigarettes. Nicotine is a key component of smoking addiction and also has some degree of damage to the human body. Studies have shown that nicotine can readily cross the blood brain barrier and biological membrane to affect the human body, especially when women are in gestation, leading to neonatal susceptibility and impaired neurological and intellectual development. With mass production and consumption of tobacco products, tobacco waste containing toxic substances such as nicotine, aminobiphenyl, naphthylamine, benzo (a) pyrene and the like enters the environment, and the toxic substances cannot be recycled, so that serious environmental problems are generated. The European Union has regulated tobacco waste with a nicotine content of over 500mg per kg as "toxic and harmful", and other countries have regulated corresponding standards. If improperly treated, these wastes can be detrimental to human health and the environment. The data show that more than 60t of tobacco waste water is required to be discharged for producing 1t of cigarettes. In recent years, as the yield is pursued on one side, the high nicotine content of flue-cured tobacco leaves in partial production areas in China is caused by applying a large amount of nitrogenous fertilizer, especially the nicotine content of upper tobacco leaves is too high to be used in cigarette formulas, and the upper tobacco leaves of 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 domestic mixed cigarettes is seriously affected. Therefore, reducing the nicotine content of tobacco leaves is one of the problems to be solved in the tobacco industry in China.
The nicotine content is one of the main indexes for determining the quality of tobacco leaves. The nicotine content of the high-quality flue-cured tobacco is generally 1.5% -3.5%, however, the nicotine content of tobacco leaves in partial areas of China is higher, especially the nicotine content of tobacco leaves at the upper part is more than 4%, so that the quality of the tobacco leaves is seriously influenced, the nerves of smokers are stimulated more strongly, and physical and mental health is further endangered. In addition, tobacco chemical composition is also important in tobacco quality. The existing tobacco chemical coordination indexes comprise sugar-egg ratio (total sugar: protein), sugar-alkali ratio (reducing sugar: nicotine), nitrogen-alkali ratio (total nitrogen: nicotine), potassium-chlorine ratio (potassium: chlorine) and oil-alkali ratio (tar: nicotine). The sugar-egg ratio is also called Shi Muke value, is used for measuring the aroma and taste of tobacco leaves, and the Shi Muke value is 2-2.5. The ratio of sugar to alkali can be measured for stiffness and alcohol and is preferably from 6 to 8. If the ratio of sugar to alkali is proper, other chemical components also tend to be balanced; the ratio of nitrogen to alkali is related to the color and the flavor of tobacco leaves, the ratio is increased, the color and the flavor are insufficient, the flavor is strong and the irritation is large if the ratio is too low, and generally 1 is preferable. The potassium-chlorine ratio is an index of combustibility, and is preferably 10 or less. The oil-alkali ratio is the smoking safety index, and is preferably below 10.
At present, the degradation of nicotine is mainly focused on physical and chemical treatment methods, so that the search for an effective and green method for degrading nicotine is an important subject in the aspect of tobacco research. The microbial degradation bacteria have the characteristics of strong degradation capability, abundant functional microorganism types, various degradation ways, small influence on the environment and the like, are a low-cost and high-efficiency nicotine treatment method, and have important significance in the aspects of reducing the content of nicotine, reducing the pollution of the nicotine, improving the utilization rate of tobacco waste, protecting water resources, protecting ecological environment and the like. Research on reduction of nicotine in tobacco leaves by microbial degradation has been started for many years at home and abroad, and has a lot of experience. At present, microbial nicotine-degrading bacteria belong to the genera Pseudomonas (Pseudomonas), arthrobacter (Acetobacter), pallidum (Palebsiella) and Agrobacterium (Agrobacterium). Brown&Williamson tobacco company utilizes Pseudomonas to degrade nicotine in tobacco, and found that after 18h treatment of mixed cut tobacco (1:1) of burley tobacco and flue-cured tobacco with Pseudomonas bacteria liquid, the nicotine content was reduced from 2.00% to 0.85% on average, resulting in a reduction of nicotine content per cigarette from 1.58mg to 0.98mg (Einosuke Wada, microbial d egradation of the tobacco alkaloids, and name related compositions of Biochemistry and biophysics.1957nov;72 (1): 145-62.). Frankburg found that microorganisms isolated from the surface of tobacco seeds degraded nicotine to formamide, ammonia, oxalic acid and trace amounts of malonic acid and succinic acid (Frankburg WG. Myoamine in cigar tobacco. Arches of biochem istry.1949Sep;23 (2): 333-5.Frankenburg WG,gottscho AM.Nicotinic acid in processed cigar tobacco.Arch Biochem.1949Mar;21 (1): 247. Frank)enburg, w.g., transformation Products of Nicotine in Fermented tobacco.science.1948.107 (2782): p.427-8.); wada separates 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 Pseudomonas (G) - ) The method comprises the steps of carrying out a first treatment on the surface of the Geiss et al isolate Nicotine-degrading Pseudomonas putida from cigar tobacco (Pseudomonas putida, G) - ) And Cellulomonas sp, which can remove nicotine and nitrate simultaneously. Wang et al isolated a highly efficient degrading strain from tobacco rhizosphere soil, which was able to completely degrade 1g/L nicotine within 6h under optimal culture conditions (Wang, S.N., Z.Liu and P.xu, biodegradation of nicotine by a newly isolatedAgrobacterium sp.strain S33.Journal of Applied Microbiology,2009.107 (3): p.838-847.); zhang Juan bacterial strain Pseudomonas marginalis ND is obtained from tobacco soil in Hunan province, and can degrade nicotine in 1g/L liquid medium by 70.40% in 2d (Zhang Juan, screening of nicotine degrading bacteria and enzyme purification and property study thereof 2012). Chen Hong et al performed a microbial enzyme degradation test for nicotine (Chen Hong et al, microbial enzyme degradation test for Total plant alkaloid of tobacco: tobacco science and technology, 2004 (4): pages 12-16), wang Ge et al separated 3 strains having a strong ability to degrade nicotine from tobacco leaves (Wang Ge, wang Yingqi, li Song et al. Microbial fermentation of tobacco leaves degraded nicotine, protein [ J ]]Tobacco science research, 2001 (2): 66.).
Domestic Chen Dexin and the like use bacillus pumilus MK21 to treat tobacco shreds, the tobacco shred nicotine degradation rate after fermentation for 5 days is 26.3%, and the aroma components of the tobacco shreds are improved. The microbial degradation of nicotine, especially the direct treatment of tobacco leaves in field period with microbe, can reduce the too high nicotine content in tobacco leaves and improve the component proportion of cigarette aroma, thus improving the utilization rate of tobacco leaf resources, and has stronger research and development value (Chen Dexin, a number of families, ma Zhiyuan, etc.. A new bacillus pumilus MK21 with high efficiency of reducing nicotine content is separated and screened and acted for research [ J ]. Chinese tobacco theory, 2013 (1): 60-64.)
Chinese patent CN113462611a discloses a kind of Arthrobacter urealyticum for degrading nicotine and its application. But the efficiency of degrading nicotine is low, and after 104 hours of culture, 2g/L of nicotine can be completely degraded. Moreover, the method is only used in an indoor experimental research stage, the field environment is more complex than the laboratory condition, the degradation efficiency is usually greatly reduced, and the strain is difficult to be applied to degrading nicotine in a true field; moreover, the bacteria only degrade nicotine, and the nicotine is only one of tobacco components, so that the bacteria also cannot guarantee the chemical coordination and various quality indexes of the tobacco, and is difficult to be applied to actual production.
In summary, the existing microorganism for degrading nicotine has low degradation efficiency and single effect, is difficult to apply in complex field environment and actual production only in indoor research stage, cannot guarantee chemical coordination and various quality indexes of tobacco, and cannot provide better use value. Therefore, it is urgently required to culture strains which degrade nicotine more efficiently, coordinate tobacco quality and exert wider application value.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide Arthrobacter urealyticum Pu17 capable of efficiently degrading nicotine and application thereof.
A first object of the present invention is to provide Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17.
A second object of the invention is to provide the use of said Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 for degrading nicotine and/or for the preparation of a biological agent for degrading nicotine.
It is a third object of the present invention to provide a biological agent for degrading nicotine.
It is a fourth object of the present invention to provide a method of degrading nicotine.
In order to achieve the above object, the present invention is realized by the following means:
a Arthrobacter urealyticus (Paenarthrobacter ureafaciens) Pu17, the Arthrobacter urealyticus (Paenarthrobacter ureafaciens) Pu17 having been deposited at the Guangdong province microbiological strain collection center under accession number GDMCC No:62557.
the application of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 in degrading nicotine and/or preparing biological agents for degrading nicotine.
A biological agent for degrading nicotine, which comprises Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 according to claim 1 or a fermentation product comprising the same.
Preferably, the biological agent contains Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of claim 1 at a concentration of 1X 10 8 ～1×10 10 cfu/mL。
More preferably, the concentration of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 is 1X 10 8 cfu/mL。
A method for degrading nicotine, using said Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 or said biological agent for degrading nicotine.
Preferably, the nicotine is the nicotine in tobacco.
More preferably, the Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 is sprayed on tobacco leaves.
More preferably, the concentration is 1X 10 8 ～1×10 10 cfu/mL or 1X 10 8 cfu/mL of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 stock solution is diluted by 14.9-15.1 times and then sprayed on tobacco leaves, and each mu of tobacco is sprayed with 0.9-1.1L of stock solution.
More preferably, the dilution factor is 15.
More preferably, 1L of the original bacterial liquid is sprayed per mu of tobacco.
More preferably, the biological agent is sprayed onto tobacco lamina.
More preferably, the Arthrobacter ureafaciens (Paenarthrobacter ureafaciens) Pu 17-containing concentration is 1X 10 8 ～1×10 10 cfu/mL or 1X 10 8 After the cfu/mL biological agent is diluted by 14.9-15.1 times, the biological agent is sprayed on tobacco leaves, and 0.9-1.1L of the biological agent is sprayed per mu of tobacco.
More preferably, the dilution factor is 15.
More preferably, 1L of the biological agent is sprayed per mu of tobacco.
More preferably, the tobacco is topping tobacco.
Compared with the prior art, the invention has the following beneficial effects:
the Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of the invention has the capability of growing with nicotine as the sole carbon and nitrogen source, and can reduce the nicotine content by 80 percent after being cultured for 30 hours on a selective medium containing 2g/L of nicotine.
Under the condition of containing other carbon and nitrogen sources, the bacteria still can preferentially utilize nicotine as a metabolic substance, has stronger nicotine metabolism capability, can reduce the nicotine content by more than 90% by culturing on an LB culture medium containing 2g/L of nicotine for 7.5 hours, can completely degrade the nicotine in 10 hours, and has high nicotine degradation efficiency.
The bacteria also has remarkable capability of degrading nicotine on tobacco plants, and the tobacco plants are sprayed with bacterial liquid containing Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 and fermentation products thereof for 24 hours, so that the nicotine content of the tobacco plants is reduced by about 30 percent; arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 is sprayed on the cured tobacco sheets, so that the smoke concentration and strength of the tobacco leaves are reduced, the comfort of the smoke is improved, and the formula applicability of the tobacco leaves is improved.
Drawings
FIG. 1 is a photograph of a plate of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 isolated on a selective medium containing nicotine. Wherein A is a selective medium containing 2g/L nicotine and B is a selective medium containing 3g/L nicotine.
FIG. 2 is a photograph of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 in LB medium containing nicotine. Wherein A is a photograph of x 1 and B is a photograph of x 7.5.
FIG. 3 is a molecular characterization phylogenetic tree of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17.
FIG. 4 shows the degradation profile of Nicotine after Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 has been cultured in a selective medium containing 2g/L of Nicotine. Wherein OD600 is the bacterial load, and peak area is the nicotine absorption peak area detected by HPLC.
FIG. 5 shows the degradation profile of nicotine after culturing Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 in LB medium containing 2 g/L. Wherein OD600 is the bacterial load, and peak area is the nicotine absorption peak area detected by HPLC.
FIG. 6 shows the nicotine content of tobacco after spraying Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 onto tobacco.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration 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, cultivation and identification of strains
1. Experimental method
1. Preparing culture medium
(1) LB medium: 10g peptone, 5g yeast extract, 10g NaCl and 15g agar were dissolved in 1L sterile ddH 2 In O water, the pH is adjusted to 7.0, and the mixture is autoclaved for 20min at 121 ℃. When the LB medium is a liquid medium, agar is not added.
(2) Preparing a microelement solution: 0.4g MnSO with 0.1mol/L HCl 4 ·7H 2 O、0.2g CaCl 2 ·2H 2 O and 0.2g FeSO 4 ·7H 2 O was dissolved and the volume was set to 100mL.
Selective medium (2 g/L): will be 13.3g K 2 HPO 4 、4g KH 2 PO 4 、0.2g MgSO 4 ·7H 2 O, 0.5mL of trace element solution and 18g of agar were mixed, pH was adjusted to 7.0, autoclaved at 121℃for 20min, cooled to 70℃and 2.2mL of 90% by mass concentration liquid nicotine was added, which was filtered through a 0.22 μm filter.
Selective medium (3 g/L): will be 13.3g K 2 HPO 4 、4g KH 2 PO 4 、0.2g MgSO 4 ·7H 2 O, 0.5mL of trace element solution and 18g of agar are mixed, the pH is adjusted to 7.0,autoclave at 121℃for 20min, cool to 70℃and add 3.3mL of liquid nicotine with a purity of 90%, which was filtered through a 0.22 μm filter.
The selective culture medium takes nicotine as the only carbon source and nitrogen source, and the nicotine concentration is 2g/L and 3g/L respectively. The selective medium is liquid medium without agar.
2. Screening nicotine degrading bacteria from soil
1.0g of soil sample from the tobacco production technology center of Yongzhou city, hunan province is weighed and added into a triangular flask with 30mL of PBS, and enrichment culture is carried out for 48h at the temperature of 30 ℃ through a shaking table at the rotating speed of 150 r/min.
Sucking 1mL of enrichment culture supernatant in an ultra-clean bench, adding the supernatant into 30mL of liquid selective culture medium, performing domestication culture for 48 hours at the rotation speed of 150r/min at the temperature of 30 ℃ by a shaking table, and separating the supernatant by a dilution plate on a nicotine solid culture medium.
The plate separation method comprises the following steps: absorbing 1mL of domesticated culture supernatant, 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 And 10 -6 Multiple bacterial liquid. Respectively taking the concentration of 10 -3 、10 -4 、10 -5 And 10 -6 0.1mL of each of the ploidy bacteria was subjected to 3 replicates for each dilution, and each culture was plated with plate-coated glass beads and incubated at 30℃for 4 days. Individual colonies were selected for further streaking and the procedure repeated until pure individual colonies were obtained and numbered.
After single colony grows out in the selective culture medium, each single colony is continuously streaked and purified for at least more than 5 times on an LB plate culture medium, then a streak plate of the isolated strain is photographed, the single colony growing out after streaking is selected by an inoculating loop is transferred into an LB liquid culture medium, bacteria are stored in a glycerol aqueous solution with volume concentration of 15% when shaking at 30 ℃ and 180rpm until the bacteria reach an exponential growth phase, and the bacteria are frozen in a refrigerator at-80 ℃ for standby. Selecting a strain with the best growth state for identification and further researching the effect of degrading nicotine.
3. Molecular biological identification
(1) The genomic DNA of the monoclonal strain of the nicotine-degrading bacterium obtained in step 2 of example 1 was extracted with a bacterial genomic DNA extraction kit (TIANamp Bacteria DNA Kit) from Tiangen biosystems, and 16S rDNA of the strain was amplified using the extracted DNA as a template and the 16S rDNA universal primers 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers.
The PCR reaction system is shown in Table 1, and after the PCR amplification system is gently mixed, the mixture is briefly centrifuged and placed on a PCR instrument according to the following steps: 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 sequencing the PCR products after detection on 1.5% agarose gel and recovery and purification by gel cutting.
TABLE 1 bacterial 16S rDNA PCR amplification System (20. Mu.L)
(2) Phylogenetic analysis of strains
Analyzing sequencing quality by using SeqMan software, splicing forward and reverse sequences, performing Blast comparison on the obtained 16S rDNA gene sequence at NCBI, selecting a strain sequence with a relatively close relationship, constructing a phylogenetic tree by using a software MEGA 7 by using a neighbor-training method, adjusting bootstrap values and checking the reliability of the evolutionary tree.
2. Experimental results
As shown in FIG. 1A, a plate with 2g/L nicotine selective medium was used to isolate photographs, as shown in FIG. 1B, a plate with 3g/L nicotine selective medium was used to isolate photographs.
As shown in FIG. 2A, a photograph of a strain isolated under a X1-fold microscope in a LB plate medium containing 2g/L nicotine, as shown in FIG. 2B, a photograph of a strain isolated under a X7.5-fold microscope in a LB plate medium containing 2g/L nicotine, a round, clean-edged opaque off-white colony formed on the LB plate by the strain, a gram-negative bacterium, a growth temperature of 30℃and a growth pH7.0; the nutrition requirement is not high, and the growth is good on the common LB culture medium and the nicotine culture medium.
The nucleotide sequence of the 16SrRNA of the purified and separated strain is shown as SEQ ID NO: 1. As shown in FIG. 3, the purified and isolated strain of the present invention has the highest similarity with Paenarthrobacter ureafaciens (NR 029281.1), its classification unit: cell organisms, the bacterial kingdom, soil bacterial groups, actinomycetes, micrococcus, arthrobacter urealyticus. The purified and isolated strain of the present invention was designated Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 and deposited at the collection of microbiological strains in Guangdong province at 2022, 6 and 20, under the accession number GDMCC No:62557 the preservation address is building 5 of No. 59 of Mitsui 100 of Guangzhou City of Guangdong province.
EXAMPLE 2 degradation of Nicotine in Selective Medium by Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17
1. Experimental method
1. Making a nicotine standard curve
Nicotine was formulated as seven concentration gradient standard solutions of 250mg/mL, 500mg/mL, 750mg/mL, 1000mg/mL, 1250mg/mL and 1500mg/mL, and a nicotine concentration standard curve was established by high performance liquid chromatography. The chromatographic column is as follows: agilentTC-C18; the mobile phase is: methanol and an aqueous solution containing 0.02mol of disodium hydrogen phosphate buffer (ph=4.2) in a volume ratio of 10:90; the column temperature is: the temperature at 30 ℃ and the flow rate are as follows: 1mL/min, the sample injection amount is as follows: 10 mu L, the detection wavelength is: 259nm.
2. 200mL of a nicotine liquid selective medium having a nicotine content of 2g/L was prepared as in example 1, and an experimental group was set up as a medium for inoculating Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of example 1, and 1% LB bacterial suspension (OD=1) was inoculated; the control group was nicotine liquid selective 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: respectively culturing for 0h, 5h, 10h, 15h, 20h, 25h and 30h in the step 2, respectively taking 2mL of bacterial liquid in an ultra-clean bench, filtering out 1mL of bacterial strain by using a filter membrane with the aperture of 0.22 mu m to obtain liquid to be detected, and detecting the nicotine content in the liquid to be detected by using high performance liquid chromatography, wherein the detection method is the same as that in the step 1; an additional 1mL of the cells were centrifuged for 2min at 1000g, resuspended in PBS and their OD600 was measured with a microplate reader at 200. Mu.L per well for 3 replicates.
2. Experimental results
As shown in FIG. 4, the nicotine content of the experimental group began to decrease at 15 hours of cultivation and 80% during 30 hours of cultivation, demonstrating that Arthrobacter ureatoberum (Paenarthrobacter ureafaciens) Pu17 of example 1 had the ability to grow on nicotine as the sole carbon and nitrogen source.
EXAMPLE 3 degradation of Nicotine in LB Medium by Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17
1. Experimental method
1. Making a standard curve
The same as in example 2.
2. Culture method
Nicotine was added to the LB medium, 200mL of LB medium having a nicotine content of 2g/L was prepared, and the preparation method of LB medium was the same as in example 1. The experimental group was set up as a medium inoculated with Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of example 1, 1% LB suspension (OD=1) was inoculated, and the control group was LB liquid medium with a nicotine content of 2g/L for the unvaccinated strain. The experimental group and the control group were cultured in an incubator at 30 ℃.
3. Detection method
Respectively culturing for 0h, 2.5h, 5h, 7.5h and 10h in the step 2, taking 2mL of bacterial liquid in an ultra-clean bench, filtering out the bacterial strain by using a filter membrane with the aperture of 0.22 mu m for 1mL to obtain a liquid to be detected, and detecting the nicotine content in the liquid to be detected by using high performance liquid chromatography, wherein the detection method is the same as that of the example 2; an additional 1mL of the cells were centrifuged for 2min at 1000g, resuspended in PBS and their OD600 was measured with a microplate reader at 200. Mu.L per well for 3 replicates.
2. Experimental results
As shown in fig. 5, the nicotine content of the experimental group is reduced by more than 50% within 5h, is reduced by more than 90% within 7.5h, and can be completely degraded within 10 h; and according to the data, the OD600 does not reach the stabilization period at 10h, which indicates that the compound has strong capability of degrading nicotine with higher concentration. It was demonstrated that Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of example 1 still preferentially utilized nicotine as a metabolic substance in the presence of other carbon and nitrogen sources, indicating that Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of example 1 has a greater nicotine metabolizing capacity.
EXAMPLE 4 degradation of Nicotine on tobacco plants by Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17
1. Experimental method
1. Culture and arrangement of bacterial cells
1L of Arthrobacter urealyticum Pu17 strain (Arthrobacter urealyticum Pu17 strain concentration 1X 10) was prepared 8 cfu/mL, since the strain is in a viable state and is growing all the time, the concentration of Arthrobacter urealyticum Pu17 strain in the bacterial liquid is 1×10 8 ～1×10 10 cfu/mL), the bacterial liquid contains Arthrobacter urealyticum Pu17 and fermentation products thereof, and is used for tobacco spraying.
2. Pu17 strain treatment method
One mu of tobacco (cloud 87) is planted until the topping period. After topping the tobacco, diluting 1L of bacterial liquid into 15L of water, and spraying the middle leaves on the tobacco. The control group was sprayed with water. After spraying, the fourth leaf of the random tobacco plant is taken at 0h, 24h, 30h, 48h and 120h respectively, and the nicotine content is detected.
3. Detection method
The fresh weight of the tobacco leaf is immediately weighed, the tobacco leaf is dried (30 min at 105 ℃ and 6h at 70 ℃), and the dry weight of the tobacco leaf is weighed after the drying. Grinding the dried tobacco leaves, and sieving 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 to obtain a tobacco activated carbon mixed solution. Heating the tobacco activated carbon mixed solution to boiling (100-101 KPa under normal pressure) by using an alcohol lamp, and keeping boiling for 5min; after cooling the solution to room temperature (25 ℃), the solution was taken up in a 250mL volumetric flask with water; after the volume is fixed, filtering, discarding 10mL of filtrate at the beginning, taking 30mL of the rest filtrate, carrying out color comparison at 236nm, 259nm and 282nm by using a spectrophotometer, measuring the light absorption value of the filtrate, and then calculating according to a formula to obtain the nicotine content. The formula is:
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
As shown in fig. 6, the experimental group had a decrease in nicotine content of about 30% at 24 hours compared to the control group, after which the nicotine content was gradually restored, and the nicotine content was still decreased by about 10% by 120 hours compared to the control group. Thus, example 1 Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 was also able to degrade nicotine on tobacco plants.
Example 5 sensory evaluation of tobacco treated with Pu17 Strain
1. Experimental method
Redried tobacco flakes of grade B2F (cloud 87) from the location of Yongzhou 2022 were treated with Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 from example 1: the Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 bacterial solution of example 1 was concentrated at a concentration of 1X 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 30min; 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.
2. Experimental results
As shown in the table 2 below,
treatment with Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of example 1 significantly improved the cloud 87 style characteristic index of the region of production in Yongzhou of 2022, reduced smoke concentration and stiffness, indicating reduced nicotine content.
In addition, according to the results, the quality characteristic index of the cloud tobacco 87 in the production area of 2022 Yongzhou is also obviously improved after the degradation treatment of the Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 in the embodiment 1. The quality characteristic index is related to nicotine, sugar content, total nitrogen content, sugar-base ratio, nitrogen-base ratio and the like.
In combination, after degradation treatment with Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of example 1, except for a significant reduction in nicotine content, the concentration and stiffness are significantly improved in style profile; but also affects other quality characteristic indexes, improves the comfort of the smoke and improves the formula applicability of the tobacco leaves. TABLE 2 Nicotine degradation treatment of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 on cloud 87-grade cured tobacco
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.
Claims (10)
1. Arthrobacter urealyticus (Paenarthrobacter ureafaciens) Pu17, wherein the Arthrobacter urealyticus (Paenarthrobacter ureafaciens) Pu17 has been deposited at the microorganism strain collection in Guangdong province at 20/6/2022 under the accession number GDMCC No:62557.
2. use of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of claim 1 for degrading nicotine and/or for the preparation of a biological agent for degrading nicotine.
3. A biological agent for degrading nicotine, comprising Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 according to claim 1 or a fermentation product thereof.
4. A biological agent for the degradation of nicotine according to claim 3, characterized in that it comprises a concentration of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 according to claim 1 of 1 x 10 8 ～1×10 10 cfu/mL。
5. The biological agent for degrading nicotine according to claim 4, wherein the concentration of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 is 1 x 10 8 cfu/mL。
6. A method for degrading nicotine, characterized in that it is degraded by the use of Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 according to claim 1 or the biological agent according to any one of claims 3 to 5.
7. The method of claim 6, wherein the nicotine is nicotine in tobacco.
8. The method of claim 7, wherein Arthrobacter urealyticum (Paenarthrobacter ureafaciens) Pu17 of claim 1 is sprayed onto tobacco leaves.
9. The method of claim 7, wherein the biological agent of any one of claims 3-5 is sprayed onto tobacco leaves.
10. The method of claim 8 or 9, wherein the tobacco is topping tobacco.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310302513.7A CN116445335A (en) | 2023-03-23 | 2023-03-23 | Arthrobacter ureafaciens Pu17 capable of efficiently degrading nicotine and application thereof |
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