CN100493359C - Sinorhizobium preparation and its application - Google Patents
Sinorhizobium preparation and its application Download PDFInfo
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- CN100493359C CN100493359C CNB2007100658800A CN200710065880A CN100493359C CN 100493359 C CN100493359 C CN 100493359C CN B2007100658800 A CNB2007100658800 A CN B2007100658800A CN 200710065880 A CN200710065880 A CN 200710065880A CN 100493359 C CN100493359 C CN 100493359C
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Abstract
The present invention relates to a kind of sinorhizobium sp. Preparation and its application. The production steps of said preparation are as follows: (a), using sinorhizobium sp.N7, CGMCC NO.1829 as production strain; (b), inoculating sinorhizobium sp. N7 into a culture liquor whose formula includes beef extract (3g/L), peptone (0.5/L), sodium chloride (0.5g/L) and the rest distilled water and whose pH value is 7.0-7.2, under the condition of that rotating speed is 150rpm and temperature is 28-30deg.C make culture for 48h 20 as to obtain liquid fermented seed; and (c), inoculating the liquid fermented seed into a fermentation tank, its liquid culture medium formula (g/L0 includes glucose 20-30, ammonium sulfate 2-5, potassium dihydrogen phosphate 0.5-1, dipotassium hydrogen phosphate 0.5-1, calcium chloride 0.1, borax 0.005, sodium molybdate 0.005 and defoaming 0.5-1, and its operation condition is as follows: fermentation temperature is 29-30deg.C, inoculation concentration is 5-10%, stirring speed is 120-150 rpm and fermentation time is 45-48hr.
Description
Technical field:
The present invention relates to a kind of Chinese nitragin preparation and application thereof, the microorganism belonging to genus technical field of pesticide.
Background technology:
Nicotine (being commonly called as nicotine) is the main component in the nicotiana alkaloids, is one of key factor that influences quality of tobacco, also is the main harmful components in tobacco industry factory effluent, the refuse.Nicotine enters behind the human body 90% and is absorbed by lung, enters blood and can arrive brain after 6 seconds, has a strong impact on the respiratory system and the sympathetic nerve of human body.In the cigarette production process, the too high levels of nicotine can increase the excitant of flue gas, influences cigarette odor-absorbing, and safety is also poor.How the nicotine content of tobacco being controlled to desired scope, is the hot subject of tobacco scientific worker and environmentalist research.The method that reduces the nicotine content in the tobacco is utilized agricultural to operate in the content of control nicotine in the tobacco growing process usually and is utilized the materialization means to reduce nicotine content in the tobacco leaf in tobacco processing course, but because above method operability is not strong, DeGrain, domestic and international research is just trending towards utilizing microbial degradation nicotine.
Microorganism particularly bacterium extensively is present under the various ecological conditions, and particular environment has bred special microbe groups.But as far back as 20th century three, the forties just relevant for the report that from soil, separates the metabolizing nicotine microorganism.Nineteen forty-seven Enders etc. just finds that saccharomycete can degrading nicotine, and the researcher has found many microorganism such as Arthrobacter globiformis (Arthrobacter globiformils) that can degrading nicotine in succession subsequently, tobacco arthrobacterium (A.nicotianae), bite nicotine arthrobacterium (A.nicotinovorans), oxidation arthrobacterium (A.oxydans), bacillus megaterium (Bacillusmegaterium), bacillus mycoides (B.mycoides), bacillus pumilus (B.pumilus), rod bacillus bacterium (Corynebacterium sp.), tobacco micrococcus luteus (Micrococcus nicotianae) etc.These microorganism majorities are by enrichment culture, get for the sole carbon nitrogenous source separates with the nicotine then.
Though the mechanism of microbial degradation nicotine also is not very clear and definite at present, the report of many using microbe live body degrading nicotines is arranged still both at home and abroad.The many solid-state or liquid waste that produces in the tobacco processing course mainly contains a large amount of alkaloids noxious materials such as nicotine, and average content reaches the 18000mg/kg dry weight.These refuses such as untimely processing bring great harm can for people's existence and life.Civilini in 1997 etc. have reported the washing lotion of oozing that adopts P.putida and A.oxidanspAO1 to handle the nicotine discarded object, and the result shows that the treatment effect of P.putida is best.Brown ﹠amp; Williamson tobacco company utilizes cellulomonas cartae to reduce nicotine and nitrate content in the tobacco.The result shows, treated burley tobaccos and other tobacco leaf are mixed, and compares with undressed burley tobaccos blade, and the nitrate content of pipe blend reduces to 1.04% from 1.63%, and nicotine reduces to 1.32% from 1.79%.After these treated pipe blends were made cigarette, flue gas analysis showed that nitrate, hydrogen cyanide and nicotine have respectively reduced by 38.8%, 19.7% and 15.3%.This shows, be of very high actual application value aborning by microorganism means reduction nicotine content and pollution abatement, its meaning not only can improve the availability of tobacco leaf resource, obtain abundant economic benefit, but also can improve the ecological environment, obtain remarkable social benefit, utilize microbial degradation nicotine to have a extensive future.
The China rhizobium are new genus of rhizobium of China scientist discovery, have obtained extensive studies and application in fields such as legume fixed nitrogen, but aspect degrading nicotine, yet there are no report at home and abroad.
Summary of the invention:
The object of the present invention is to provide a kind of nicotine degradation rate height, and the Chinese nitragin preparation and the application thereof that can improve cigarette quality.
The present invention is achieved in that
1, the acquisition of Chinese rhizobium (Sinorhizobium sp.) N7
Be selected from tobacco leaf, tobacco sample grinds, and the dilution spread plate is at M9+ nicotine medium: Na
2HPO
412.8g, KH
2PO
43.0g, NaCl 0.5g, NH
4Cl 0.5g, agar powder 17g, nicotine 10g (sterilization separately), 10.0g vitamin B1 (sterilization separately) 1 μ g/ml, pH nature, under 28 ℃ of temperature, cultivate 2d, picking can be on flat board the bacterial clump of healthy growth, bacterium colony performance is wherein arranged better, name bacterial strain into N7.
The N7 bacterial strain is carried out biology and biochemical test, Gram-negative bacteria.Shaft-like, 0.6～0.8 μ m * 1.2～3.0 μ m.Do not produce gemma.1～3 of flagellum Zhousheng.Aerobic.On the NA medium bacterium colony rounded, protruding, mucus arranged, yeast extract-mannitol-mineral salt agar after last 3 day diameter be generally 2～3mm.Optimum growth temperature is 25～35 ℃, and this bacterial strain can be grown at 38 ℃ and 10 ℃; Optimal pH 6.0～8.0.Can utilize following material as sole carbon source: glucose, D-arabinose, cellobiose, fructose, D-galactose, D-mannose, mannitol, L-ancient acid amides, lactose, D-ribose, succinate, wood sugar and D-turanose.Can not utilize following carbon source: winestone acid amide, oxamide, cellulose, defend that hair alcohol, rock are sugared, glycine, sorbose, alginic acid sodium and vanillic acid.On YMA medium, produce acid.Can grow in 1.0%NaCl.Can utilize ammonium chloride and nitrate as carbon source, utilize the peptone ability.Oxidase and catalase are positive.
This bacterium is carried out the 16SrDNA sequencing, the GenBank number of registration:
DQ659139N7 is accredited as certain kind (Sinorhizobium sp.) of Chinese rhizobium by sequence alignment and biochemical characteristic.
Chinese rhizobium of the present invention (Sinorhizobium sp.) N7 on October 9th, 2006 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, address: No. 13, ZhongGuanCun north, Haidian District, BeiJing City bar, deposit number is CGMCC NO.1829, classification called after: Chinese rhizobium (Sinorhizobium sp).
2, prepare Chinese nitragin preparation,
The ratio of using among the present invention is g/L.
A. slant culture: medium is a nutrient agar, 28～30 ℃ of heat insulating culture 28h.
B. shake-flask seed is cultivated: Chinese rhizobium N7 slant strains is inoculated into prescription in beef extract 3g/L, peptone 0.5g/L, sodium chloride 0.5g/L, all the other culture fluids for distilled water, pH 7.0-7.2, shaking bottle rotating speed is 150rpm, under 28-30 ℃ of temperature, cultivate 48h, obtain the liquid fermentation seed.
C. liquid fermentation process: fermentation seed is inoculated into liquid culture basigamy ten thousand (g/L) is housed: glucose 20～30; Sulfate of ammoniac 2～5; Potassium dihydrogen phosphate 0.5～1, dipotassium hydrogen phosphate 0.5～1, calcium chloride 0.1, borax 0.005, sodium molybdate 0.005, defoamer 0.8, all the other are in the fermentation tank of water, under 29-30 ℃ of fermentation temperatures, inoculum concentration 5～10%, fermentation tank mixing speed 120-150rpm, throughput 5-10%, 45-48 hours condition of fermentation time, obtain Chinese nitragin preparation, put into container and keep in Dark Place, finish using in 1 week.Wherein, inoculum concentration and throughput ratio adopt percent by volume.
Chinese nitragin preparation of the present invention is as the application of the nicotine in degrading tobacco or the tobacco waste.
Chinese nitragin preparation of the present invention has nicotine degradation rate height, and can improve advantages such as cigarette quality.
Description of drawings:
Accompanying drawing is growth and the nicotine degradation design sketch (implement 1) of N7 bacterial strain in containing the liquid of nicotine.
Embodiment:
Embodiment 1:(is used to contain nicotine liquid)
One, prepare Chinese nitragin preparation:
The bacterial classification that preparation adopts is Chinese rhizobium (Sinorhizobium sp.) N7 bacterial strains.
A. fermented bacterium is cultivated: Chinese rhizobium N7 slant strains is inoculated into prescription (g/L) in beef extract 3, peptone 0.5, sodium chloride 0.5, all the other culture fluids for distilled water, pH7.0, shaking bottle rotating speed is 150rpm, under 28 ℃ of temperature, cultivated 48 hours, obtain the liquid fermentation seed.
B. ferment tank: the test tube kind is inoculated into liquid culture based formulas (g/L) is housed: glucose 20; Sulfate of ammoniac 2; Potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate 0.5, calcium chloride 0.1, borax 0.005, sodium molybdate 0.005, defoamer 0.5, all the other are in the fermentation tank of water, in 29 ℃ of fermentation temperatures, inoculum concentration 5%.Obtain Chinese nitragin preparation under fermentation tank mixing speed 120rpm, throughput 5%, 45 hours the condition of fermentation time.Wherein, inoculum concentration and throughput ratio adopt percent by volume.
Two, the application of Chinese nitragin preparation:
Nornicotine is degraded preparation inoculation (inoculum concentration 1%) in the M9 liquid that contains 0.1% nicotine, and 30 ℃ of 150rpm medicine bottles are cultivated, took a sample every 2 hours nicotine content that detects solution and the solution 0D600 value of representing thalline quantity.Application result is seen accompanying drawing, degrades that nicotine content is 0.014% after 24 hours, promptly use preparation with the nicotine degradation in the solution 86%.
Embodiment 2:(is used to contain nicotine liquid)
Substantially with embodiment 1.Difference is: during the test tube kind is cultivated: pH is 7.2,30 ℃ of temperature; In the zymotechnique: glucose 30; Sulfate of ammoniac 5; Potassium dihydrogen phosphate 1, dipotassium hydrogen phosphate 1, defoamer 1,30 ℃ of fermentation temperatures, inoculum concentration 10%.Fermentation tank mixing speed 150rpm, throughput 10%, fermentation time 48 hours.
Embodiment 3:(is used for cured tobacco leaf to be handled)
The preparation of China's nitragin preparation is substantially with embodiment 1.Difference is: during fermented bacterium is cultivated: pH is 7.1,29 ℃ of temperature.In the zymotechnique: fermentation seed is inoculated into the liquid culture based formulas is housed: glucose 28, sulfate of ammoniac 3, potassium dihydrogen phosphate 0.8, dipotassium hydrogen phosphate 0.8, defoamer 0.8; 29.5 ℃ of fermentation temperatures, inoculum concentration 8%.Fermentation tank mixing speed 130rpm, throughput 8%, fermentation time 46 hours.
The application of nicotine degradation preparation of the present invention:, put into long * wide * height and be the cement pit of 1.5m * 0.5m * 1m with 10 times of preparation dilute with waters of the present invention.Compile bar behind the tobacco leaf picking that maturation is good.Tobacco leaf behind the volume bar is put into the pond of containing microbial inoculum and is soaked evenly, takes out, and spontaneous current loses excessive solution, and in the barn of packing into then, baking method routinely toasts.Tobacco leaf after the baking carries out the detection of nicotine, and the result shows that nicotine degradation preparation of the present invention can reduce by 22.6% flue-cured tobacco nicotine.
Use the tobacco leaf aesthetic quality smoking result of the flue-cured tobacco behind the preparation of the present invention to show: the strength of tobacco leaf obviously reduces, and fragrance matter and perfume quantity have in various degree to be improved; Excitant obviously weakens, and assorted gas obviously alleviates.
Claims (2)
1, a kind of Chinese nitragin preparation obtains behind fermentation seed cultivation, ferment tank by producing bacterial strain, it is characterized in that said preparation is obtained by the following step:
A. the production bacterial strain of selecting for use is Chinese rhizobium (Sinorhizobium sp.) N7, CGMCC NO.1829;
B. fermentation seed is cultivated: Chinese rhizobium N7 slant strains is inoculated into prescription in beef extract 3g/L, peptone 0.5g/L, sodium chloride 0.5g/L, all the other culture fluids for distilled water, pH7.0-7.2, shaking bottle rotating speed is 150rpm, under 28-30 ℃ of temperature, cultivate 48h, obtain the liquid fermentation seed;
C. ferment tank: the liquid fermentation seed is inoculated into the fermentation cylinder for fermentation that the liquid fermentation system is housed; Culture medium prescription is: glucose 20～30g/L, sulfate of ammoniac 2～5g/L, potassium dihydrogen phosphate 0.5～1g/L, dipotassium hydrogen phosphate 0.5～1g/L, calcium chloride 0.1g/L, borax 0.005g/L, sodium molybdate 0.005g/L, defoamer 0.5～1g/L; Operating condition is: 29-30 ℃ of fermentation temperatures, and inoculum concentration 5～10%, fermentation tank mixing speed 120-150rpm, throughput 5-10%, fermentation time are 45-48 hours; Wherein, inoculum concentration and throughput ratio adopt percent by volume.
2, the application of the described Chinese nitragin preparation of claim 1 is characterized in that the application of said preparation as the nicotine in degrading tobacco or the tobacco waste.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433276B (en) * | 2011-11-30 | 2013-04-03 | 云南大学 | Sinorhizobium sp. and application thereof |
| CN103275888B (en) * | 2013-04-09 | 2014-11-05 | 南京师范大学 | Ensifer meliloti and its use in degradation of chloronicotinyl insecticide thiacloprid |
| CN110813078A (en) * | 2019-11-26 | 2020-02-21 | 许达勇 | Nicotine decomposer and preparation method thereof |
| CN110903875A (en) * | 2019-12-06 | 2020-03-24 | 龙门县景龙生物能源有限公司 | Biomass granular fuel produced by utilizing tobacco waste and preparation method thereof |
Citations (3)
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|---|---|---|---|---|
| CN1330716A (en) * | 1998-11-09 | 2002-01-09 | G.In.E.St.R.A.有限合伙公司 | Method for control gene expression in bacterial, namely i(rhizobiaceae) to improved root nodule development, nitrogen fixation and plant blomass production |
| WO2005062899A2 (en) * | 2003-12-23 | 2005-07-14 | Nitragin, Inc. | Methods and compositions providing agronomically benefical effects in legumes and non-legumes |
| CN1951197A (en) * | 2006-11-01 | 2007-04-25 | 沈阳农业大学 | A novel method for controlling soybean root disease by using rhizobia |
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2007
- 2007-05-16 CN CNB2007100658800A patent/CN100493359C/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1330716A (en) * | 1998-11-09 | 2002-01-09 | G.In.E.St.R.A.有限合伙公司 | Method for control gene expression in bacterial, namely i(rhizobiaceae) to improved root nodule development, nitrogen fixation and plant blomass production |
| WO2005062899A2 (en) * | 2003-12-23 | 2005-07-14 | Nitragin, Inc. | Methods and compositions providing agronomically benefical effects in legumes and non-legumes |
| CN1951197A (en) * | 2006-11-01 | 2007-04-25 | 沈阳农业大学 | A novel method for controlling soybean root disease by using rhizobia |
Non-Patent Citations (2)
| Title |
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| 苜蓿中华根瘤菌与耐盐有关的DNA片段的克隆. 陈雪松等.微生物学报，第39卷第06期. 1999 |
| 苜蓿中华根瘤菌与耐盐有关的DNA片段的克隆. 陈雪松等.微生物学报，第39卷第06期. 1999 * |
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