CN104263688A - Arthrobacter sp SCUEC3 strain as well as screening method and application thereof - Google Patents
Arthrobacter sp SCUEC3 strain as well as screening method and application thereof Download PDFInfo
- Publication number
- CN104263688A CN104263688A CN201410524617.3A CN201410524617A CN104263688A CN 104263688 A CN104263688 A CN 104263688A CN 201410524617 A CN201410524617 A CN 201410524617A CN 104263688 A CN104263688 A CN 104263688A
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- Prior art keywords
- nicotine
- bacterial strain
- scuec3
- arthrobacter
- strain
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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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- 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
- C12R2001/06—Arthrobacter
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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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- 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
Abstract
The invention provides an arthrobacter sp SCUEC3 strain. The preservation number of the strain is CCTCC NO: M2014402. A nucleotide sequence of the strain is shown as SEQ ID NO.1. The strain can be applied to nicotine degradation and can be used for accelerating maturing of tobacco leaves. Meanwhile, the invention also provides a method for screening and separating the strain. The method comprises the following steps: firstly, performing enrichment culture; secondly, performing separation and purification; finally, inoculating the separated and purified strain in a nicotine culture medium according to a single colony, performing shake cultivation at room temperature for 1-3 days, determining the content of nicotine in the fermentation liquor of the nicotine culture medium, and re-screening the strain according to the size of the nicotine degradation rate to obtain the arthrobacter sp SCUEC3 strain.
Description
Technical field
The present invention relates to a kind of microorganism strains of degrading nicotine, be specifically related to a kind of Arthrobacter SCUEC3 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 Arthrobacter SCUEC3 bacterial strain, can not only applying in nicotine degradation of this bacterial strain, but also tobacco leaf maturation can be accelerated.
Arthrobacter SCUEC3 provided by the invention (Arthrobacter sp SCUEC3) bacterial strain, the deposit number of this bacterial strain is: CCTCC NO:M2014402, 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 Arthrobacter SCUEC3 (Arthrobacter sp SCUEC3) 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%.
Arthrobacter SCUEC3 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 90.08% in the lab, and in field test, the nicotine degradation rate of this bacterial strain reaches as high as 26.13%.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.
Arthrobacter SCUEC3 (the Arthrobacter sp SCUEC3) 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:M2014402, the 16S nucleotide sequence of this bacterial strain, as shown in SEQ ID NO.1, comprises 1177 bases.
The method for screening and separating of Arthrobacter SCUEC3 (the Arthrobacter sp SCUEC3) 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 Arthrobacter SCUEC3 (Arthrobacter sp SCUEC3) 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 Arthrobacter SCUEC3 (the Arthrobacter sp SCUEC3) 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 compound nitrogen source, the concentration of trace element for 5mL/L, under this optimal conditions, nicotine degradation rate can reach 90.08%.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 Arthrobacter SCUEC3 (the Arthrobacter sp SCUEC3) 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 26.13%.Meanwhile, find that Arthrobacter SCUEC3 (Arthrobacter sp SCUEC3) bacterial strain can also accelerate tobacco leaf maturation through test.
Claims (6)
1., for Arthrobacter SCUEC3 (Arthrobacter sp SCUEC3) bacterial strain for nicotine degradation, the deposit number of this bacterial strain is: CCTCC NO:M2014402, 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 Arthrobacter SCUEC3 (Arthrobacter sp SCUEC3) 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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| CN201410524617.3A CN104263688A (en) | 2014-09-30 | 2014-09-30 | Arthrobacter sp SCUEC3 strain as well as screening method and application thereof |
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| CN201410524617.3A CN104263688A (en) | 2014-09-30 | 2014-09-30 | Arthrobacter sp SCUEC3 strain as well as screening method and application thereof |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525579A (en) * | 2009-04-09 | 2009-09-09 | 浙江中烟工业有限责任公司 | Pseudomonad ZCJ bacterial strain applied to nicotine degradation of tobacco and screening method and application thereof |
-
2014
- 2014-09-30 CN CN201410524617.3A patent/CN104263688A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525579A (en) * | 2009-04-09 | 2009-09-09 | 浙江中烟工业有限责任公司 | Pseudomonad ZCJ bacterial strain applied to nicotine degradation of tobacco and screening method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 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》 * |
| 孔雯等: "1株烟碱降解菌的筛选、鉴定及其降解性能的初步研究", 《华中农业大学学报》 * |
| 李天丽等: "烟叶醇化过程中烟碱降解菌的分离鉴定与特性分析", 《烟草科技》 * |
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