CN102643772B - Microbe strain and application thereof - Google Patents
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- CN102643772B CN102643772B CN201210142423.8A CN201210142423A CN102643772B CN 102643772 B CN102643772 B CN 102643772B CN 201210142423 A CN201210142423 A CN 201210142423A CN 102643772 B CN102643772 B CN 102643772B
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Abstract
The invention discloses a microbe strain and an application thereof and particuarly relates to a tobacco smoke alkaloid degradation bacterium (Arthrobacter nicotianae GYC103 with the collection number of CCTCC NO:M2010312) capable of simultaneously generating amylolytic enzyme and proteolytic enzyme and an application of the tobacco smoke alkaloid degradation bacterium to tobacco. The strain disclosed by the invention can decompose and utilize nicotine and generate amylolytic enzyme and proteolytic enzyme in a growth process. A strain fermentation liquid or thallus accounting for 1-5wt% of tobacco leaves is added into the tobacco leaves with the water content of 10-50%, and fermentation is performed for 6-72 hours, so that the nicotine content of the tobacco leaves is reduced by 2-20%, the starch content of the tobacco leaves is reduced by 10-35%, the protein content of the tobacco leaves is reduced by 5-25%, the pungency of the tobacco leaves is obviously reduced, the miscellaneous flavor is reduced, the smoke is soft, the tobacco fragrance is enhanced, and the esthetic quality is obviously improved. The microbe strain realizes the purpose of degrading tobacco smoke alkaloid, starch and protein by utilizing microbes, and can be used for properly regulating the contents of nicotine, starch and protein in tobacco leaf raw materials and improving the usability of the tobacco leaves.
Description
Technical field
The present invention relates to strain microorganism strains and an application thereof, be specifically related to a kind of can hold concurrently produce amylase and proteolytic enzyme tobacco smoke alkaloid degradation bacteria---tobacco Arthrobacter (Arthrobacter nicotianae GYC103) CCTCC NO:M2010312 and the application in tobacco thereof, belong to technical field of microbe application.
Background technology
Nicotine has another name called Nicotine (Nicotine), is the main component in nicotiana alkaloids.In flue-cured tobacco, the content of nicotine, generally 1.5%～3.5%, is advisable with 2.5% left and right.Nicotine content is too low, and strength is too little, and nicotine content is too high, and strength is too large, can cause and sting the pungent sense of choking.China some areas tobacco leaf visual appearance approaches world level in recent years, but tobacco leaf chemical composition not too coordinates, and nicotine is higher, especially upper tobacco leaf.Reduce the nicotine content in tobacco, mainly contain at present three approach: (1) regulates and controls from the angle of agricultural planting: mainly from aspects such as heredity, ecology and cultivations, control.(2) from chemical angle: the alkaloid tobacco leaf can be by taking off the nicotine in tobacco leaf by the method for the processing tobacco leaves such as hot water rinsing, organic solvent extraction, gas extracting and vapor distillation.(3) from the angle of microorganism and enzyme: from tobacco leaf, cigarette seed and separated microorganism that can degrading nicotine soil, directly or after isolating enzyme system act on tobacco leaf after cultivating, reduce the nicotine content in tobacco leaf, thereby improve the operability of tobacco leaf.For ripe tobacco leaf of having gathered, just can only adopt the 2nd and 3 kinds of methods.Wherein, although use the chemical processes such as solvent extraction can remove a part of nicotine, can cause the loss of some aroma components in tobacco and the noticeable change of appearance luster simultaneously, thereby reduce to a certain extent the operability of processing tobacco leaf.And process tobacco leaf by microorganism or enzyme, because enzyme has specificity, therefore can avoid preferably these problems.
Utilize biotechnological formulation to process tobacco leaf and can shorten fermentation time, regulation and control tobacco leaf objectionable constituent, as nicotine and TSNA etc., promote macromolecular material as the conversion of protein and starch etc., promote tabacco fragrance.Aspect microbiological deterioration nicotine, C.Enders etc. were just studied yeast degradation nicotine as far back as nineteen forty-seven, became gradually the problem that people pay close attention to later.More external large tobacco enterprises, as Philips Maurice tobacco company, British American Tobacco etc. utilize microorganism to degrade to the nicotine in tobacco very early, meet the demand of a part of consumer group to low Nicotine cigarette with this.Reported at present can degrading nicotine microorganism mainly comprise 2 large classes: yeast and bacterium, abroad report as Deharyomyces nicotianae, Micrococusnicotianae, Pseudomonas, Alcaligenes paradox us, Arthrobacterglobif ormils, Enterobacter cloacae, Cunninghamella echinulata, Nicotianae plumbaginif olia, Arthrobacter oxidans etc.The nicotine degradation approach of having reported has three: (1) tetramethyleneimine approach (pyrrolidine pathway); (2) pyridine approach (pyridine pathway); (3) demethylation approach (Me pathway).The tobacco pseudomonas (Pseudomonas nicotianae) that domestic Sun Jun society etc. (2002) isolate degrading nicotine from tobacco leaf warehouse or outdoor cigarette buttress soil around, but only propose that degrading nicotine is had to effect.
In tobacco leaf, except nicotine, also has a large amount of starch and protein.The carbohydrate existing with starch form can affect combustionvelocity and combustion completion when cigarette burns and sucks, and during its burning, produces and sticks with paste burnt flavour, can make tobacco flavor degenerate.Protein is the basic material that forms cell, its hydrolysate and the product further transforming are the hyles of many tobacco aroma materials, but protein content is high, can produce a kind of protein stink as burning feather while burning and sucking, reduce the incendivity of tobacco leaf, and be the precursor of harmful substances from flue gases, comprise Kui Lin, HCN and other nitrogenous compounds, have a strong impact on fragrance quality and the security of tobacco leaf.If can screen, obtaining a strain can degrading nicotine, and the microorganism of can degrade again tobacco leaf starch and protein, to utilizing biotechnology to improve especially upper smoke quality of China's tobacco leaf, improves tobacco leaf usability and have important effect.
Summary of the invention
Main purpose of the present invention is to provide a kind of microorganism strains that can degrading nicotine simultaneously produces amylase and proteolytic enzyme to be tobacco Arthrobacter Arthrobacter nicotianae GYC103 (preserving number is CCTCC NO:M2010312) and corresponding culture medium and culture condition and disclose a kind of method of utilizing nicotine, protein and starch raising tobacco leaf usability in microbiological deterioration tobacco.
The present invention is achieved by the following scheme:
A microorganism strains, is characterized in that: it is tobacco Arthrobacter Arthrobacter nicotianae GYC103, and preserving number is CCTCC NO:M2010312; Gram-positive, anaerobism is negative, and catalase is positive, oxidase negative, gelatine liquefication is negative, and nitrate reduction is positive, and Citrate trianion is positive, urease negative, malonate is negative, and V-P is negative, and salt tolerance is positive; Glucose is negative, and N.F,USP MANNITOL is negative, and lactose is negative, and sucrose is negative, and maltose is negative, and wood sugar is negative, and ribose is negative; The yellow bacterium colony of smooth opaque, the neat in edge of visible surface while growing on substratum, optimum growth temperature is 25-30 ℃.
Described bacterial strain obtains by following approach:
First from soil or tobacco leaf, isolate the microorganism strains of degrading nicotine;
Secondly with milk, select substratum to screen the microorganism strains of acquisition, acquisition can produce the bacterial strain of proteolytic enzyme;
Then with starch, select Screening of Media can produce diastatic bacterial strain the bacterial strain obtaining, acquisition is the nicotine degradation bacterium of degrade proteins and starch simultaneously, i.e. tobacco Arthrobacter Arthrobacter nicotianae GYC103.
The culturing step of described bacterial strain is:
A, slant culture: peptone 10g, yeast extract paste 5g, sodium-chlor 10g, agar 15g, distilled water 1000ml, pH7.0, by the glycerine suspension inoculation of freezing preservation to slant medium, 30 ℃ of constant temperature culture 2 days;
B, shake-flask culture: nicotine 2g, peptone 5g, yeast extract paste 5g, sodium-chlor 5g, K
2hPO
43H
2o13.3g, KH
2pO
44g, MgSO
47H
2o0.2g, 0.5ml trace element, adding distil water 1000ml, adjust pH is 7.0, high pressure steam sterilization; Shaking flask liquid amount is 10～30v/v%, and inoculum size is 2～10 v/v%, and culture temperature is 20～40 ℃, shaking speed is 100～200rpm, shake-flask culture 1～3 day, and nicotine degradation rate reaches 85～100%, proteinase activity reaches 18～60U/ml, and diastatic activity reaches 10～40U/ml.
Described trace element is: CaCl
20.04g, CuSO
40.07g, MnSO
4h
2o0.008g, FeSO
47H
2o0.004g, Na
2moO
42H
2o0.1g, adds 0.1molL
-1hCl dissolve constant volume 100ml.
Described microorganism strains can degrading nicotine, in process of growth, can produce amylase and proteolytic enzyme.
Described tobacco Arthrobacter Arthrobacter nicotianae GYC103 is for nicotine, starch and the protein of degrading tobacco; The said nicotine for degrading tobacco, starch and protein, obtain as follows:
A, tobacco Arthrobacter Arthrobacter nicotianae GYC103 claimed in claim 1 is carried out to fermentation culture, obtain fermented liquid and thalline;
B, the fermented liquid of step a gained or thalline water, physiological saline or damping fluid are diluted, after dilution, bacterial concentration reaches 10
6～10
12individual bacterium/ml, according to 1～5% of tobacco leaf weight, joins bacterium liquid water content and is in 10～50% tobacco and ferment, and leavening temperature is 10～50 ℃, and fermentation time is greater than 4 hours.
Wherein in steps A, said fermented liquid is any one in barbitol buffer solution, phthalate buffer, ammonia-ammonium chloride buffer, borax-calcium chloride damping fluid, acetic acid-sodium-acetate buffer, acetic acid-ammonium acetate buffer, citrate buffer or phosphate buffered saline buffer.
Fermented liquid after cultivation bacterial concentration after dilution reaches 10
9individual/ml, according to 1～5% of tobacco leaf weight, joins bacterium liquid water content and is in 10～50% tobacco, ferment 6～72 hours, can make tobacco leaf nicotine, protein and starch content reduce, pungency obviously reduces, flue gas is soft, and cigarette is fragrant to be increased, and tobacco leaf usability obviously improves.
Described tobacco Arthrobacter Arthrobacter nicotianae GYC103, is deposited in Chinese Typical Representative culture collection center on November 24th, 2010, and it is referred to as CCTCC, and deposit number is CCTCC NO:M2010312.Preservation address: Luojiashan, Wuchang, Wuhan City, Hubei Province, postcode 430072.
Compared with prior art, beneficial effect of the present invention is:
Adopt the present invention to improve the especially quality of upper tobacco leaf of raw tobacco material, technological process is simple, and reaction conditions is gentle; After treatment, tobacco leaf nicotine, protein and starch content reduce, and pungency obviously reduces, and flue gas is soft, and cigarette is fragrant to be increased, and tobacco leaf usability obviously improves.The features such as the method has simple and practical, with low cost, are expected to apply industrial.
Figure of description
For ease of understanding tobacco Arthrobacter molecular biological characteristics of the present invention, spy is described further by reference to the accompanying drawings.
Fig. 1 is the 16SrDNA sequence chart of tobacco Arthrobacter.
Embodiment
Below will by embodiment, the invention will be further elaborated, its objective is as better and understand content of the present invention.Therefore, the cited case does not limit the scope of the invention.
Embodiment 1
Slant culture: peptone 10g, yeast extract paste 5g, sodium-chlor 10g, agar 15g, distilled water 1000ml, pH7.0, by the glycerine suspension inoculation of freezing preservation to slant medium, 30 ℃ of constant temperature culture 2 days.
Shake-flask culture: nicotine 1g, peptone 5g, yeast extract paste 5g, sodium-chlor 5g, K
2hPO
43H
2o13.3g, KH
2pO
44g, MgSO
47H
2o0.2g, 0.5ml trace element, adding distil water 1000ml, adjust pH is 7.0, high pressure steam sterilization.250ml shaking flask liquid amount is 30ml, and inoculum size is 3v/v%, and culture temperature is 30 ℃, and shaking speed is 200rpm, shake-flask culture 36 hours, and nicotine degradation rate reaches 100%, and proteinase activity reaches 28U/ml, and diastatic activity reaches 36U/ml.Trace element: CaCl
20.04g, CuSO
40.07g, MnSO
4h
2o0.008g, FeSO
47H
2o0.004g, Na
2moO
42H
2o0.1g, adds 0.1molL
-1hCl dissolve constant volume 100ml.
Amylase enzyme activity (U) definition: under 45 ℃, pH7.0 condition, be hydrolyzed the required enzyme amount of 1mg starch in every 10 minutes.
Proteinase activity power (U) definition: under 37 ℃, pH7.0 condition, per minute caseinhydrolysate produces the required enzyme amount of 1 μ g tyrosine.
Embodiment 2
Slant culture is identical with embodiment 1.In 250ml shaking flask, pack 30ml substratum into, it is composed as follows: nicotine 2g, corn steep liquor 15g, yeast extract paste 5g, sodium-chlor 5g, K
2hPO
43H
2o13.3g, KH
2pO
44g, MgSO
47H
2o0.2g, 0.5ml trace element, adding distil water 1000ml, adjust pH is 7.0, high pressure steam sterilization.250ml shaking flask liquid amount is 30ml, and inoculum size is 2v/v%, and culture temperature is 30 ℃, and shaking speed is 200rpm, shake-flask culture 48 hours, and nicotine degradation rate reaches 100%, and proteinase activity reaches 54U/ml, and diastatic activity reaches 28U/ml.Trace element is identical with embodiment 1.The definition of amylase enzyme activity and proteinase activity power is identical with embodiment 1.
Embodiment 3
Get the fermented liquid 10ml in embodiment 1, with sterile distilled water, be diluted to 10
9individual bacterium/ml, by bacterium liquid according to 1% of tobacco leaf weight, spray application to water content and be in Shaoyang upper tobacco leaf B2F in 2008 of 18%, 35 ℃, the condition bottom fermentation of relative humidity 75% 10 hours, can make that nicotine content of tobacco leaves declines 2.9%, protein content declines 5.6%, starch content reduces by 10.8%, tobacco leaf pungency obviously reduces, assorted gas reduces, and flue gas is soft, and cigarette is fragrant to be increased.
Embodiment 4
Get the fermented liquid 10ml in embodiment 1, with sterile distilled water, be diluted to 10
9individual bacterium/ml, by bacterium liquid according to 1% of tobacco leaf weight, spray application to water content and be in Shaoyang upper tobacco leaf B2F in 2008 of 18%, 35 ℃, the condition bottom fermentation of relative humidity 75% 36 hours, can make that nicotine content of tobacco leaves declines 9.1%, protein content declines 12.2%, starch content reduces by 13.6%, tobacco leaf pungency obviously reduces, assorted gas reduces, and flue gas is soft, and cigarette is fragrant to be increased.
Embodiment 5
Get the fermented liquid 10ml in embodiment 1, with sterile distilled water, be diluted to 10
9individual bacterium/ml, by bacterium liquid according to 1% of tobacco leaf weight, spray application to water content and be in Shaoyang upper tobacco leaf B2F in 2008 of 18%, 35 ℃, the condition bottom fermentation of relative humidity 75% 48 hours, can make that nicotine content of tobacco leaves declines 12%, protein content declines 18.5%, starch content reduces by 28.8%, tobacco leaf pungency obviously reduces, assorted gas reduces, and flue gas is soft, and cigarette is fragrant to be increased.
Embodiment 6
Get the fermented liquid 10ml in embodiment 1, with sterile distilled water, be diluted to 10
9individual bacterium/ml, by bacterium liquid according to 2% of tobacco leaf weight, spray application to water content and be in Shaoyang upper tobacco leaf B2F in 2008 of 20%, 35 ℃, the condition bottom fermentation of relative humidity 75% 36 hours, can make that nicotine content of tobacco leaves declines 11.2%, protein content declines 21.6%, starch content reduces by 31.3%, tobacco leaf pungency obviously reduces, assorted gas reduces, and flue gas is soft, and cigarette is fragrant to be increased.
Embodiment 7
Get the fermented liquid 10ml in embodiment 1, with sterile distilled water, be diluted to 10
9individual bacterium/ml, by bacterium liquid according to 2% of tobacco leaf weight, spray application to water content and be in Shaoyang upper tobacco leaf B2F in 2008 of 20%, 35 ℃, the condition bottom fermentation of relative humidity 75% 48 hours, can make that nicotine content of tobacco leaves declines 18.8%, protein content declines 25.3%, starch content reduces by 35.2%, tobacco leaf pungency obviously reduces, assorted gas reduces, and flue gas is soft, and cigarette is fragrant to be increased.
Claims (7)
1. microorganism strains, is characterized in that: it is tobacco Arthrobacter (Arthrobacter nicotianae) GYC103, and preserving number is CCTCC NO:M2010312.
2. microorganism strains as claimed in claim 1, is characterized in that: described bacterial strain is Gram-positive, and anaerobism is negative, and catalase is positive, oxidase negative, gelatine liquefication is negative, and nitrate reduction is positive, and Citrate trianion is positive, urease negative, malonate is negative, and V-P is negative, and salt tolerance is positive; Glucose is negative, and N.F,USP MANNITOL is negative, and lactose is negative, and sucrose is negative, and maltose is negative, and wood sugar is negative, and ribose is negative; The yellow bacterium colony of smooth opaque, the neat in edge of visible surface while growing on substratum, optimum growth temperature is 25-30 ℃.
3. microorganism strains as claimed in claim 1 or 2, is characterized in that, the culturing step of described bacterial strain is:
A, slant culture: peptone 10g, yeast extract paste 5g, sodium-chlor 10g, agar 15g, distilled water 1000ml, pH7.0, by the glycerine suspension inoculation of freezing preservation to slant medium, 30 ℃ of constant temperature culture 2 days;
B, shake-flask culture: nicotine 2g, peptone 5g, yeast extract paste 5g, sodium-chlor 5g, K
2hPO
43H
2o13.3g, KH
2pO
44g, MgSO
47H
2o0.2g, 0.5ml trace element, adding distil water 1000ml, adjust pH is 7.0, high pressure steam sterilization; Shaking flask liquid amount is 10～30v/v%, and inoculum size is 2～10v/v%, and culture temperature is 25～30 ℃, shaking speed is 100～200rpm, shake-flask culture 1～3 day, and nicotine degradation rate reaches 85～100%, proteinase activity reaches 18～60U/ml, and amylase activity reaches 10～40U/ml;
Wherein, described trace element is: CaCl
20.04g, CuSO
40.07g, MnSO
4h
2o0.008g, FeSO
47H
2o0.004g, Na
2moO
42H
2o.1molL O0.1g, add
-1hCl dissolve constant volume 100ml.
4. microorganism strains as claimed in claim 1 or 2, is characterized in that: it can degrading nicotine, in process of growth, can produce amylase and proteolytic enzyme.
5. microorganism strains as claimed in claim 3, is characterized in that: it can degrading nicotine, in process of growth, can produce amylase and proteolytic enzyme.
6. the application of tobacco Arthrobacter GYC103 as described in claim 1, is characterized in that, said tobacco Arthrobacter GYC103 is for nicotine, starch and the protein of degrading tobacco; Step is as follows:
A, tobacco Arthrobacter (Arthrobacter nicotianae) GYC103 is carried out to fermentation culture, obtain fermented liquid and thalline;
B, the fermented liquid of steps A gained or thalline water, physiological saline or damping fluid are diluted, after dilution, bacterial concentration reaches 10
6～10
12individual bacterium/ml, according to 1～5% of tobacco leaf weight, joins bacterium liquid water content and is in 10～50% tobacco and ferment, and leavening temperature is 35～50 ℃, and fermentation time is greater than 4 hours.
7. application as claimed in claim 6, it is characterized in that, wherein in step B, said damping fluid is any one in barbitol buffer solution, phthalate buffer, ammonia-ammonium chloride buffer, borax-calcium chloride damping fluid, acetic acid-sodium-acetate buffer, acetic acid-ammonium acetate buffer, citrate buffer or phosphate buffered saline buffer.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103373867B (en) * | 2013-06-24 | 2015-09-23 | 湖南碧野农业科技开发有限责任公司 | The biochemical treatment of tobacco invalid body and Fertilizer Transformed utilize technique |
| CN103642722B (en) * | 2013-11-27 | 2015-06-24 | 湖北省烟草公司恩施州公司 | Nicotine-degrading bacterium and application thereof |
| CN103789238A (en) * | 2014-01-27 | 2014-05-14 | 华中农业大学 | Nicotine efficient degrading bacterium and culture method |
| CN104164390B (en) * | 2014-07-09 | 2016-07-06 | 青岛蔚蓝天成生物科技有限公司 | One strain nicotianae and the application in aquaculture thereof |
| CN104388376B (en) * | 2014-11-07 | 2017-06-30 | 河南省烟草公司漯河市公司 | A kind of nicotine degradation bacterium screening and culturing medium and preparation method thereof |
| CN109161569B (en) * | 2018-09-21 | 2021-07-16 | 郑州轻工业学院 | Method for producing bacterial cellulose by fermenting tobacco extracts through microorganism combination |
| CN110195027B (en) * | 2019-03-08 | 2022-03-01 | 大连理工大学 | Tobacco arthrobacter ZL-1 and preparation method and application of compost low-temperature fermentation microbial inoculum thereof |
| CN113773980B (en) * | 2021-07-31 | 2023-02-17 | 广东中烟工业有限责任公司 | Enterobacter nicotianae NLB1 for degrading nicotine and application thereof |
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