CN101177669A - Crude enzyme liquid prepared by microorganism and uses thereof - Google Patents
Crude enzyme liquid prepared by microorganism and uses thereof Download PDFInfo
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- CN101177669A CN101177669A CNA2007100659165A CN200710065916A CN101177669A CN 101177669 A CN101177669 A CN 101177669A CN A2007100659165 A CNA2007100659165 A CN A2007100659165A CN 200710065916 A CN200710065916 A CN 200710065916A CN 101177669 A CN101177669 A CN 101177669A
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- tobacco
- nicotine
- crude enzyme
- microorganism
- pseudomonas
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Abstract
The invention relates to a crude enzyme produced by microorganism and an application thereof, pertaining to the microorganism application technical field. The invention is obtained by producing strain and auxiliary material through liquid enlarging culture and liquid fermentation culture; the producing strain is pseudomonas sp which is preserved in a common microorganism center of China Committee for Culture Collection of microorganisms on April 12, 2007, and the preservation number is: CGMCC No.1998. The crude enzyme prepared by the microorganism of the invention can be used for reducing the content of the nicotine in tobacco. The invention has the advantages of being capable of adjusting the nicotine content in tobacco leaf material, enhancing the usability of tobacco life and degrading the nicotine in the smoke and reducing the pollution to environment, etc.
Description
Technical field:
The present invention relates to a kind of crude enzyme liquid and application thereof, belong to the using microbe technical field with the microorganism preparation.
Background technology:
Tobacco is one of mainstay of China's finance, along with China becomes a full member of WTO and signature WHO " Framework Convention on Tobacco Control ", the internationalization level in China's cigarette and tobacco leaf market is progressively deepened, " Framework Convention on Tobacco Control " is strict further to the restriction of tobacco product, and Chinese tobacco is faced with new situation and new challenge.For this reason, State Tobacco Monopoly Bureau has formulated " Chinese cigarette development in science and technology outline ", and the direction that clearly proposes the Chinese cigarette development is: market-oriented, greatly develop the Chinese style cigarette.And the gordian technique of development Chinese style cigarette is exactly will be under the prerequisite that keeps high fragrance " reducing tar and reducing harm ", the Chinese style cigarette brand of exploitation " low tar, low harm, high fragrance ".Therefore, reducing the content of objectionable constituent such as nicotine in the tobacco, is to promote one of cigarette industry key of healthy development technology.
Nicotine is the main component in the nicotiana alkaloids, and in tobacco, the nicotine major part is to exist with the state of organic acid (as citric acid and oxysuccinic acid) salt, also has a spot ofly to exist with free state.Nicotine enters in the human body, and 90% is absorbed by lung, enter blood after 6s can arrive brain.The most significant effect is that sympathetic nerve is exerted an influence to nicotine to human body, is usually expressed as of short duration excitement, and then is exactly to suppress.The effect of nicotine mainly also is the physiological strength that it produced except increasing smoke and impression stimulation, be commonly referred to " strength ".In general, the tobacco leaf that nicotine content is high, flue gas strength is big, otherwise then little.Therefore, it is necessary containing a certain amount of nicotine in the flue gas, but the content of nicotine can not be too high, otherwise not only can increase the pungency of flue gas, influences cigarette odor-absorbing, and also is a very adverse factors from the angle of security.
The tobacco leaf visual appearance in China part cigarette district is near world level, but also has certain gap with regard to its inner quality, and one of them is general and be exactly that nicotine content is higher than distinct issues.At present, the average content of China's flue-cured tobacco nicotine is 3%～4%, and burley tobaccos are on average up to 6%, and the nicotine content of U.S.'s flue-cured tobacco is between 1.5%～3.5%; The nicotine content of general high-quality burley tobaccos is between 1.5%～4.0%, and 3.0% to be good, the nicotine content of U.S.'s burley tobaccos is about 2.9%.The upper tobacco leaf that domestic cigarette enterprise is stored at present is more, generally is difficult to the more tobacco leaf formulation that is used for because of its nicotine content is higher, has not only taken a large amount of storage spaces, and has caused a large amount of wastes.This problem has caused the attention of the parties concerned, and the nicotine content that how to reduce in these tobacco leaves is the realistic problem that each cigarette enterprise faces.Because the existence of the continuous cultivation of tobacco and a large amount of waste tobacco leafs improves the nicotine content in the soil greatly, edatope and groundwater quality have been caused bigger influence simultaneously.
About having report by degrading nicotine (Nicotine) with multiple microorganism.But by literature search, find open report with content same document of the present invention.
Summary of the invention:
The objective of the invention is to research, a kind of method that reduces nicotine content in the tobacco is provided by the microorganism pseudomonas nic22 (Pseudomonas sp.nic22) of nicotine (Nicotine) content in can degrading tobacco to a strain.
The present invention has separated the bacterium nic22 that a strain has the degrading nicotine ability from tobacco cultivation soil, it has been carried out morphology, Physiology and biochemistry character and 16S rRNA analyzes, qualification result shows that this bacterium belongs to Pseudomonas, this bacterial strain called after pseudomonas (Pseudomonas sp.).This bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on April 12nd, 2007; Preserving number is CGMCC No.1998.
Main morphological specificity and the Physiology and biochemistry character of pseudomonas nic22 (Pseudomonas sp.nic22) are:
The Nic22 thalline is rod-short, the blunt circle in two ends, no gemma.Gram-negative, no pod membrane, amphimicrobian.Utilize nitrate, urea and ammonium salt.The Nic22 bacterial strain can produce catalase, oxydase, and the indoles that can not liquefy does not produce hydrogen sulfide.Methyl red (MR) experiment and acetyl methyl carbinol (VP) experiment are all negative.The Nic22 bacterial strain can utilize sucrose, glucose, starch, maltose and seminose, can not utilize Mierocrystalline cellulose, pectin, and it is vigorous to grow in the solid medium that with glucose, nicotine or seminose is sole carbon source, and bacterium colony is flat, oyster white, the edge is neat.Be in the substratum of sole carbon nitrogenous source with nicotine, Nic22 produces pigment, cultivate 2d after, bacterium colony by green redden brown.
The Molecular Identification of pseudomonas nic22 (Pseudomonas sp.nic22) is characterized as: utilize universal primer 16F (5-AGAGTTTGATCCTGGCTCAG-3) and 1500R (5-GGTTACCTTGTTACGACT-3), obtain PCR product about 1500bp with the genomic dna of bacterium nic22 as template amplification, the 16S rRNA sequence of the bacterial strain Px6-4 that reclaim order-checking, obtains after proofreading and correct.The 16s rRNA nucleotide sequence total length 1498bp of nic22 bacterial strain, this sequence has been submitted international nucleotide sequence database (GenBank), and sequence index number is: EF541115.
The present invention prepares according to a conventional method.Be about to obtain after pseudomonas nic22 (Pseudomonas sp.nic22) enlarges fermentation culture.
Crude enzyme liquid of the present invention is as the application that reduces nicotine content in the tobacco.
The present invention has the nicotine content that can adjust raw tobacco material, improves tobacco leaf usability, and can decompose the nicotine in the flue dust, reduces its advantage such as pollution to environment.
Embodiment:
Below be embodiments of the invention, but content of the present invention is not limited thereto.
The present invention is achieved in that
The cultural method of pseudomonas nic22 (Pseudomonas sp.nic22):
1. the test tube slant is cultivated
Adopt Luria-Bertani (LB) substratum, filling a prescription is: 1% peptone, 1% sodium-chlor, 0.5% yeast extract, 1.8-2% agar.Sterilized 25 minutes, and put into the inclined-plane for 121 ℃.Inoculation Pseudomonas sp.nic22 cultivated 18-24 hour for 37 ℃, obtained the test tube kind.
2. fluid enlargement culture
1) seed culture (LB substratum), filling a prescription is: 1% peptone, 1% sodium-chlor, 0.5% yeast extract.Sterilized 25 minutes for 121 ℃.Inoculation Pseudomonas sp.nic22 cultivated 18-24 hour for 30 ℃, obtained liquid spawn.
2) fermention medium: 1mL tobacco leaf water extract, 1g KH
2PO
4, 0.5g MgSO
4, 1mL MnSO
4(0.1%), 3mL FeSO
4(0.1%), 3mL KCl (1%) adds water to 1000mL, pH6～7.The initial pH7.0 of substratum, inoculum size 3-5%, rotating speed 180rpm, 30 ℃ of temperature.Cultivated 48 hours at this fermentation condition bottom fermentation.
2. the preparation of pseudomonas nic22 (Pseudomonas sp.nic22) nornicotine crude enzyme liquid
Utilize fermention medium to prepare the fermented liquid of 1000mL, the centrifugal 15min of 8000rpm collects thalline, damping fluid (the 50mM phosphate buffered saline buffer of thalline with sterilization, pH7.0) suspend, the centrifugal 15min of 8000rpm collects thalline, repeat twice, somatic cells suspends with the damping fluid of 50ml at last.The somatic cells suspension liquid carries out break process with ultrasonic wave, and the centrifugal 15min of 8000rpm removes broken somatic cells, and supernatant liquor (crude enzyme liquid)-20 ℃ preservation is standby.
3. the nornicotine of pseudomonas nic22 (Pseudomonas sp.nic22) nornicotine crude enzyme liquid experiment
Take by weighing 50g upper tobacco leaf Yuxi B respectively
3The F pipe tobacco, according to certain addition (be respectively tobacco quality 2%, 5%, 10%) with crude enzyme liquid evenly spray be added on the pipe tobacco, control pipe tobacco relative humidity is 20%, places climatic chamber to preserve 2 days for 30 ℃ again.
With the pipe tobacco without the crude enzyme liquid processing is contrast, compares with three groups of pipe tobaccos of handling through different addition crude enzyme liquids and smokes panel test.Simultaneously, adopt the tobacco automatic chemical analyzer to measure the contrast pipe tobacco and handle nicotine content in the pipe tobacco, every sample do three times parallel, get average, analytical results sees Table 1.
The result of table 1:Pseudomonas sp.nic22 crude enzyme liquid degrading nicotine
| Sample | Crude enzyme liquid dosage (%) | Nicotine content (%) | Relative nicotine content (%) |
| Contrast | 0 | 3.54 | 100 |
| Sample 1 | 2 | 3.07 | 86.7 |
| Sample 2 | 5 | 2.69 | 75.9 |
| Sample 3 | 10 | 2.37 | 66.9 |
Claims (2)
1. crude enzyme liquid with microorganism preparation, obtain after fluid enlargement culture, liquid fermentation and culture by producing bacterial strain and auxiliary material, it is characterized in that this bacterial strain is pseudomonas (Pseudomonas sp.), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 12nd, 2007, preserving number is CGMCC No.1998.
2. the application of the described crude enzyme liquid with microorganism preparation of claim 1 is characterized in that this crude enzyme liquid is as the application that reduces nicotine content in the tobacco.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100659165A CN101177669B (en) | 2007-05-29 | 2007-05-29 | Crude enzyme liquid prepared by microorganism and uses thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100659165A CN101177669B (en) | 2007-05-29 | 2007-05-29 | Crude enzyme liquid prepared by microorganism and uses thereof |
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| Publication Number | Publication Date |
|---|---|
| CN101177669A true CN101177669A (en) | 2008-05-14 |
| CN101177669B CN101177669B (en) | 2010-04-14 |
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|---|---|---|---|
| CN2007100659165A Expired - Fee Related CN101177669B (en) | 2007-05-29 | 2007-05-29 | Crude enzyme liquid prepared by microorganism and uses thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102258213A (en) * | 2011-02-25 | 2011-11-30 | 福州农博士生物技术有限公司 | Complex enzyme bacterium preparation used for tobacco thread production and preparation method thereof |
| CN102433276A (en) * | 2011-11-30 | 2012-05-02 | 云南大学 | Sinorhizobium sp. and application thereof |
| CN102732440A (en) * | 2011-04-12 | 2012-10-17 | 华中农业大学 | Pseudomonas putida T2-2 able to reduce nitrosamines in flue-cured tobacco leaves and application thereof |
| US10405571B2 (en) | 2015-06-26 | 2019-09-10 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having altered alkaloid levels |
| CN112226477A (en) * | 2020-10-13 | 2021-01-15 | 深圳市太丰东方海洋生物科技有限公司 | Preparation method and application of novel marine shellfish micromolecular immunoactive peptide |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1180078C (en) * | 2002-07-03 | 2004-12-15 | 中国农业大学 | Microbe for degrading tobacco nicotine, and method for separating and culturing same and use thereof |
-
2007
- 2007-05-29 CN CN2007100659165A patent/CN101177669B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102258213A (en) * | 2011-02-25 | 2011-11-30 | 福州农博士生物技术有限公司 | Complex enzyme bacterium preparation used for tobacco thread production and preparation method thereof |
| CN102258213B (en) * | 2011-02-25 | 2013-07-17 | 福州农博士生物技术有限公司 | Complex enzyme bacterium preparation used for tobacco thread production and preparation method thereof |
| CN102732440A (en) * | 2011-04-12 | 2012-10-17 | 华中农业大学 | Pseudomonas putida T2-2 able to reduce nitrosamines in flue-cured tobacco leaves and application thereof |
| CN102433276A (en) * | 2011-11-30 | 2012-05-02 | 云南大学 | Sinorhizobium sp. and application thereof |
| US10405571B2 (en) | 2015-06-26 | 2019-09-10 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having altered alkaloid levels |
| CN112226477A (en) * | 2020-10-13 | 2021-01-15 | 深圳市太丰东方海洋生物科技有限公司 | Preparation method and application of novel marine shellfish micromolecular immunoactive peptide |
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| Publication number | Publication date |
|---|---|
| CN101177669B (en) | 2010-04-14 |
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