CN104328067A - Ochrobactrum intermedium SCUEC4 strain, screening method and uses thereof - Google Patents
Ochrobactrum intermedium SCUEC4 strain, screening method and uses thereof Download PDFInfo
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- CN104328067A CN104328067A CN201410522701.1A CN201410522701A CN104328067A CN 104328067 A CN104328067 A CN 104328067A CN 201410522701 A CN201410522701 A CN 201410522701A CN 104328067 A CN104328067 A CN 104328067A
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- nicotine
- bacterial strain
- scuec4
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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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
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/26—Organic substances containing nitrogen or phosphorus
Abstract
The present invention provides an Ochrobactrum intermedium SCUEC4 strain, wherein the preservation number is CCTCC NO:M2014403, the nucleotide sequence of the strain is represented by SEQ ID NO.1, and the strain can be applied in nicotine degradation and can further be provided for accelerating tobacco leaf maturation. The present invention further provides a method for screening and separating the strain, wherein the method comprises: carrying out enrichment culture, carrying out separation purification, inoculating the separated and purified single colony into a nicotine culture medium, carrying out shaking culture for 1-3 days at a room temperature, determining the nicotine content in the fermentation broth of the nicotine culture medium, and re-screening the strain according to the nicotine degradation rate so as to obtain the Ochrobactrum intermedium SCUEC4 strain.
Description
Technical field
The present invention relates to a kind of microorganism strains of degrading nicotine, be specifically related to a kind of in the middle of anthropi SCUEC4 bacterial strain, and the screening method of this bacterial strain and purposes, belong to nicotine degradation technical field.
Background technology
Nicotine is not only the most important chemical composition be present in tobacco, and the quality of height to tobacco product of nicotine content plays considerable effect.At present, China's Flue-cured Tobacco alkali content is generally higher, especially upper tobacco leaf is more obvious, as: subregion, Shennongjia surrounding area tobacco leaf visual appearance is close to internal and international advanced level, but some Chan Yan county nicotine content of tobacco leaves are up to 3.5% ~ 4.5%, and burley tobaccos upper smoke alkali content is especially up to 6%.This not only have impact on the quality of upper smoke, and affects the operability on top, brings difficulty to cigarette composition industry, constrains upper tobacco leaf market management.
The nicotine content effectively reduced in high-nicotine content tobacco leaf is the realistic problem that tobacco industry faces rapidly, thus, large quantity research is done to the nicotine content reduced in high-nicotine content tobacco leaf both at home and abroad, these researchs mostly concentrate on ecology, cultural facts, physical measure reduction nicotine content, as fertilising, filtration, punching dilution, tobacco sheet etc., and less to utilizing biotechnology degrading nicotine to study report.
(application number is Chinese invention patent application: 200710013068.3,200710013069.8,200710070805.3,200910097536.9, Deng), the bacterial strain that these patents relate to has effective degradation property to nicotine, and unique shortcoming carries out nicotine degradation under indoor laboratory condition, whether has effect also confirm without experiment for the true field tobacco leaf produced.
Summary of the invention
The invention provides a kind of middle anthropi SCUEC4 bacterial strain, can not only applying in nicotine degradation of this bacterial strain, but also tobacco leaf maturation can be accelerated.
Middle anthropi SCUEC4 provided by the invention (Ochrobactrum intermedium SCUEC4) bacterial strain, the deposit number of this bacterial strain is: CCTCC NO:M2014403, and the nucleotide sequence of this bacterial strain is as shown in SEQ ID NO.1.
The present invention additionally provides a kind of method of screening and separating above-mentioned bacterial strains simultaneously, comprises the following steps:
(1), enrichment culture: the soil in tobacco planting field is put into nicotine substratum, shaking table is cultivated at ambient temperature, cultivate after 1-3 days, pregnant solution is forwarded in fresh nicotine substratum and continues to cultivate, repeat to cultivate relaying step more than three times, to reach the object of enrichment, collect enrichment culture liquid for subsequent use;
(2), separation and purification: adopt physiological saline by gradient stepwise dilution enrichment culture liquid, then diluent is coated in nicotine isolation medium, cultivates, then choose fast growth, bacterial strain that the growth impetus is good carries out separation and purification;
(3), the bacterial strain after separation and purification is pressed single colony inoculation in nicotine substratum, at room temperature shaking table is cultivated after 1-3 days, measure nicotine content in the fermented liquid of nicotine substratum, according to the size reflex sieve bacterial strain of nicotine degradation rate, thus obtain middle anthropi SCUEC4 (Ochrobactrum intermedium SCUEC4) bacterial strain.
Described culturing step is specially: under 30 DEG C of conditions, and adopt 180rpm shaking table to cultivate, incubation time is 48h.
Described physiological saline is the physiological saline of mass concentration 0.9%.
Middle anthropi SCUEC4 bacterial strain provided by the invention has the following advantages: 1, the degradation capability of this bacterial strain is strong, after tested, the degradation rate of this bacterial strain can reach 92.68% in the lab, and in field test, the nicotine degradation rate of this bacterial strain reaches as high as 27.34%.2, this bacterial strain can accelerate the maturation of tobacco leaf.
Accompanying drawing explanation
Fig. 1 is nicotine degradation rate curve table in the embodiment of the present invention.
Embodiment
Below in conjunction with specific embodiment, detailed specific description is done to the present invention, but protection scope of the present invention is not limited to following examples.
Middle anthropi SCUEC4 (the Ochrobactrum intermedium SCUEC4) bacterial strain provided in the present embodiment is preserved in China typical culture collection center on September 9th, 2014, address is: Wuchang District, Wuhan City, Hubei Province Wuhan University, deposit number is CCTCC NO:M2014403, the 16S nucleotide sequence of this bacterial strain, as shown in SEQ IDNO.1, comprises 1160 bases.
The method for screening and separating of middle anthropi SCUEC4 (the Ochrobactrum intermedium SCUEC4) bacterial strain provided in the present embodiment is as follows: (1), enrichment culture: the soil 2g in tobacco planting field is put into nicotine substratum 20mL, under 30 DEG C of conditions, adopt 180rpm shaking table to cultivate, after cultivating 48h, 1mL pregnant solution is forwarded in fresh nicotine substratum and continues to cultivate, repeat to cultivate relaying step three times, to reach the object of enrichment, collect enrichment culture liquid for subsequent use;
(2), separation and purification: adopt the physiological saline of mass concentration 0.9% by gradient stepwise dilution enrichment culture liquid, then diluent is coated in nicotine isolation medium, cultivate, then choose fast growth, the good bacterial strain of the growth impetus carries out separation and purification;
In this step because nicotine isolation medium is using nicotine as sole carbon source, nitrogenous source is inorganic salt, and the microorganism that therefore can grow on this isolation medium is exactly the microorganism of the ability may with degrading nicotine.
(3), the bacterial strain after separation and purification is pressed single colony inoculation in nicotine substratum, at room temperature shaking table is cultivated after 1-3 days, measure nicotine content in the fermented liquid of nicotine substratum, according to the size reflex sieve bacterial strain of nicotine degradation rate, thus obtain middle anthropi SCUEC4 (Ochrobactrum intermedium SCUEC4) bacterial strain.
Although the bacterial strain that can grow on above nicotine isolation medium likely has the ability of degrading nicotine, also likely just resistance to nicotine does not degrade it, so will carry out multiple sieve to the bacterium colony of purifying in this step.
The experiment of laboratory smoke alkaline degradation is carried out by screening middle anthropi SCUEC4 (the Ochrobactrum intermedium SCUEC4) bacterial strain obtained in the present embodiment, determine that optimum degradation condition is by analysis afterwards: nicotine starting point concentration is 2g/L, temperature is 30 DEG C, initial pH is 5.5-6.0, using peptone and extractum carnis each 0.5% as the concentration of compound nitrogen source, trace element for 5mL/L, under this optimal conditions, nicotine degradation rate can reach 92.68%.Experimental result as shown in Figure 1, can be found out in FIG, and along with the growth of incubation time, nicotinic density reduces gradually, and absorbancy constantly raises.
Then field nicotine degradation experiment is carried out, bacteria suspension is made by screening middle anthropi SCUEC4 (the Ochrobactrum intermedium SCUEC4) bacterial strain obtained in the present embodiment, fresh tobacco leaves before bacteria suspension process is gathered, after having tested, effectively can reduce nicotine content in tobacco leaf after measured, nicotine degradation rate is 27.34%.Meanwhile, in the middle of test finds, anthropi SCUEC4 (Ochrobactrum intermedium SCUEC4) bacterial strain can also accelerate tobacco leaf maturation.
Claims (6)
1. the middle anthropi SCUEC4 for nicotine degradation (Ochrobactrum intermedium SCUEC4) bacterial strain, the deposit number of this bacterial strain is: CCTCC NO:M2014403, and the nucleotide sequence of this bacterial strain is as shown in SEQ ID NO.1.
2. a method for screening and separating for bacterial strain described in claim 1, is characterized in that comprising the following steps:
(1), enrichment culture: the soil in tobacco planting field is put into nicotine substratum, shaking table is cultivated at ambient temperature, cultivate after 1-3 days, pregnant solution is forwarded in fresh nicotine substratum and continues to cultivate, repeat to cultivate relaying step more than three times, to reach the object of enrichment, collect enrichment culture liquid for subsequent use;
(2), separation and purification: adopt physiological saline by gradient stepwise dilution enrichment culture liquid, then diluent is coated in nicotine isolation medium, cultivates, then choose fast growth, bacterial strain that the growth impetus is good carries out separation and purification;
(3), the bacterial strain after separation and purification is pressed single colony inoculation in nicotine substratum, at room temperature shaking table is cultivated after 1-3 days, measure nicotine content in the fermented liquid of nicotine substratum, according to the size reflex sieve bacterial strain of nicotine degradation rate, thus obtain middle anthropi SCUEC4 (Ochrobactrum intermedium SCUEC4) bacterial strain.
3. method for screening and separating according to claim 2, is characterized in that: described culturing step is specially: under 30 DEG C of conditions, and adopt 180rpm shaking table to cultivate, incubation time is 48h.
4. method for screening and separating according to claim 2, is characterized in that: described physiological saline is the physiological saline of mass concentration 0.9%.
5. the purposes of bacterial strain according to claim 1 in nicotine degradation.
6. bacterial strain according to claim 1 is accelerating the purposes in tobacco leaf maturation.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410522701.1A CN104328067A (en) | 2014-09-30 | 2014-09-30 | Ochrobactrum intermedium SCUEC4 strain, screening method and uses thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112336A (en) * | 2015-09-06 | 2015-12-02 | 中国农业科学院烟草研究所 | Ochrobactrum intermedium CGMCC No.10670 tolerant to high-concentration nicotine and application thereof |
| CN111909874A (en) * | 2020-08-17 | 2020-11-10 | 东北农业大学 | Biocontrol bacterium I-5 and application thereof in controlling alfalfa root rot |
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| US4037609A (en) * | 1975-11-17 | 1977-07-26 | Brown & Williamson Tobacco Corporation | Process for reduction of nicotine content of tobacco by microbial treatment |
| CN1800361A (en) * | 2005-12-06 | 2006-07-12 | 南京农业大学 | Method of nicotine biological degradation |
| CN101016528A (en) * | 2007-01-22 | 2007-08-15 | 山东大学 | Pseudomonas putida capable of metabolizing nicotine and application thereof |
| CN103992973A (en) * | 2014-05-22 | 2014-08-20 | 中南民族大学 | Agrobacterium tumefaciens SCUEC1 strain as well as screening method and application thereof |
-
2014
- 2014-09-30 CN CN201410522701.1A patent/CN104328067A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US4037609A (en) * | 1975-11-17 | 1977-07-26 | Brown & Williamson Tobacco Corporation | Process for reduction of nicotine content of tobacco by microbial treatment |
| CN1800361A (en) * | 2005-12-06 | 2006-07-12 | 南京农业大学 | Method of nicotine biological degradation |
| CN101016528A (en) * | 2007-01-22 | 2007-08-15 | 山东大学 | Pseudomonas putida capable of metabolizing nicotine and application thereof |
| CN103992973A (en) * | 2014-05-22 | 2014-08-20 | 中南民族大学 | Agrobacterium tumefaciens SCUEC1 strain as well as screening method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| YUAN YJ等: "Isolation and preliminary characterization of a novel nicotine-degrading bacterium, Ochrobactrum intermedium DN2", 《INTERNATIONAL BIODETERIORATION & BIODEGRADATION》 * |
| 孔雯: "烟碱降解菌的分离鉴定、降解特性及其降解途径的初步研究", 《中国优秀硕士论文全文数据库 农业科技辑》 * |
| 张彦东等: "微生物降解烟碱研究应用进展", 《烟草科技／烟草工艺》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112336A (en) * | 2015-09-06 | 2015-12-02 | 中国农业科学院烟草研究所 | Ochrobactrum intermedium CGMCC No.10670 tolerant to high-concentration nicotine and application thereof |
| CN105112336B (en) * | 2015-09-06 | 2018-11-13 | 中国农业科学院烟草研究所 | Anthropi CGMCC No.10670 and its application in the base of enduring high-concentration nicotine |
| CN111909874A (en) * | 2020-08-17 | 2020-11-10 | 东北农业大学 | Biocontrol bacterium I-5 and application thereof in controlling alfalfa root rot |
| CN111909874B (en) * | 2020-08-17 | 2022-08-19 | 东北农业大学 | Biocontrol bacterium I-5 and application thereof in controlling alfalfa root rot |
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Application publication date: 20150204 |