CN102643772A - Microbe strain and application thereof - Google Patents
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- CN102643772A CN102643772A CN2012101424238A CN201210142423A CN102643772A CN 102643772 A CN102643772 A CN 102643772A CN 2012101424238 A CN2012101424238 A CN 2012101424238A CN 201210142423 A CN201210142423 A CN 201210142423A CN 102643772 A CN102643772 A CN 102643772A
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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 a strain microorganism strains and an application thereof; Being specifically related to a kind of can holding concurrently and producing the tobacco smoke alkaloid degradation bacteria of glycase and proteolytic enzyme---tobacco Arthrobacter (Arthrobacter nicotianae GYC103) CCTCC NO:M2010312 and in Application in Tobacco belongs to technical field of microbe application.
Background technology
Nicotine has another name called Nicotine (Nicotine), is the staple in the nicotiana alkaloids.The content of nicotine is advisable with about 2.5% generally 1.5%～3.5% in the flue-cured tobacco.Nicotine content is low excessively, and strength is too little, and nicotine content is too high, and strength is too big, can cause to sting the pungent sense of choking.China some areas tobacco leaf visual appearance is near 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 the tobacco, mainly contain three approach at present: (1) is regulated and control from the angle of agricultural planting: mainly control from aspects such as heredity, ecology and cultivations.(2) from the angle of chemistry: the vegeto-alkali the tobacco leaf can be through taking off the nicotine in the tobacco leaf with the method for processing tobacco leaves such as hot water rinsing, organic solvent extraction, gas extracting and vapor distillation.(3) from the angle of mikrobe and enzyme: from tobacco leaf, cigarette seed and soil, separate can degrading nicotine mikrobe, cultivate the back and directly or after isolating enzyme system act on tobacco leaf, reduce the nicotine content in the tobacco leaf, thereby improve the operability of tobacco leaf.For ripe tobacco leaf of having gathered, just can only adopt the 2nd and 3 kind of method.Wherein, though chemical processes such as use solvent extraction can be removed a part of nicotine, can cause the loss of some aroma components in the tobacco and the noticeable change of appearance luster simultaneously, thereby reduce the operability of handling tobacco leaf to a certain extent.And handle tobacco leaf through mikrobe or enzyme, because enzyme has specificity, therefore can avoid these problems preferably.
Utilize biotechnological formulation to handle tobacco leaf and can shorten fermentation time, regulation and control tobacco leaf objectionable constituent such as nicotine and TSNA etc. promote the conversion of macromolecular material such as protein and starch etc., promote tabacco fragrance.Aspect microbiological deterioration nicotine, C.Enders etc. just studied yeast degradation nicotine as far back as nineteen forty-seven, became the problem that people pay close attention to later on gradually.More external big tobacco enterprises such as Philips Maurice tobacco company, British American Tobacco etc. utilize mikrobe that the nicotine in the tobacco is degraded very early, satisfy the demand of a part of consumer group to low Nicotine cigarette with this.Reported at present can degrading nicotine mikrobe mainly comprise 2 big types: yeast and bacterium, abroad report like 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) of isolating degrading nicotine soil around tobacco leaf warehouse or the open-air cigarette buttress such as domestic Sun Jun society (2002), but only propose that degrading nicotine is had effect.
Except nicotine, also has a large amount of starch and protein in the tobacco leaf.The carbohydrate that exists with the starch form props up when burning and sucking at cigarette and can influence combustionvelocity and combustion completion, produces during its burning and sticks with paste burnt flavour, and tobacco flavor is degenerated.Protein then is the basic material that constitutes cell; Its hydrolysate is the hyle of many tobacco aroma materials with the product that further transforms; But can produce a kind of protein stink when the protein contnt height burns and sucks, reduce the incendivity of tobacco leaf, and be the precursor of objectionable impurities in the flue gas as the burning feather; Comprise Kui Lin, HCN and other nitrogenous compounds, have a strong impact on the fragrance quality and the security of tobacco leaf.Can degrading nicotine if can screen acquisition one strain, can degrade again tobacco leaf starch and proteinic mikrobe are improved especially upper smoke quality of China's tobacco leaf to utilizing biotechnology, and the raising tobacco leaf usability has important effect.
Summary of the invention
Main purpose of the present invention is to provide a kind of microorganism strains that can degrading nicotine produces glycase and proteolytic enzyme simultaneously to be tobacco Arthrobacter Arthrobacter nicotianae GYC103 (preserving number is CCTCC NO:M2010312) and to produce the enzyme substratum accordingly and culture condition and disclose a kind of method of utilizing nicotine, protein and starch raising tobacco leaf usability in the microbiological deterioration tobacco.
The present invention realizes through following scheme:
A kind of 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 SANMALT-S is negative, and wood sugar is negative, and ribose is negative; The yellow bacterium colony of smooth opaque, the neat in edge of when growth visible surface on substratum, optimum growth temperature is 25-30 ℃.
Said bacterial strain obtains through following approach:
At first from soil or tobacco leaf, isolate the microorganism strains of degrading nicotine;
Secondly select substratum to screen with milk the microorganism strains that obtains, acquisition can produce the bacterial strain of proteolytic enzyme;
Select screening of medium can produce diastatic bacterial strain with starch the bacterial strain that obtains then, acquisition is the nicotine degradation bacterium of degrade proteins and starch simultaneously, i.e. tobacco Arthrobacter Arthrobacter nicotianae GYC103.
The culturing step of said bacterial strain is:
A, slant culture: peptone 10g, yeast extract paste 5g, sodium-chlor 10g, agar 15g, zero(ppm) water 1000ml, pH7.0, with 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, the 0.5ml trace element, adding distil water 1000ml, adjust pH are 7.0, high pressure steam sterilization; Shaking bottled liquid measure 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 the nicotine degradation rate reaches 85～100%; Proteinase activity reaches 18～60U/ml, and diastatic activity reaches 10～40U/ml.
Said 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 dissolving constant volume 100ml.
Said microorganism strains can degrading nicotine, in process of growth, can produce glycase and proteolytic enzyme.
Said tobacco Arthrobacter Arthrobacter nicotianae GYC103 is used for nicotine, starch and the protein of degrading tobacco; Said nicotine, starch and the protein that is used for degrading tobacco obtains through following steps:
A, the described tobacco Arthrobacter of claim 1 Arthrobacter nicotianae GYC103 is carried out fermentation culture, obtain fermented liquid and thalline;
B, fermented liquid or thalline water, saline water or the damping fluid of step a gained diluted, dilution back bacterial concentration reaches 10
6～10
12Individual bacterium/ml according to 1～5% of tobacco leaf weight, joins bacterium liquid water cut and is in 10～50% the tobacco and ferment, and leavening temperature is 10～50 ℃, and fermentation time was greater than 4 hours.
Wherein said fermented liquid is 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 the phosphate buffered saline buffer any one in the steps A.
Fermented liquid after cultivation bacterial concentration after diluting reaches 10
9Individual/ml, bacterium liquid according to 1～5% of tobacco leaf weight, is joined water cut and is in 10～50% the tobacco; Fermented 6～72 hours, tobacco leaf nicotine, protein and starch content are reduced, pungency obviously reduces; Flue gas is soft, and cigarette is fragrant to be increased, and tobacco leaf usability obviously improves.
Said tobacco Arthrobacter Arthrobacter nicotianae GYC103 is deposited in Chinese typical culture collection center on November 24th, 2010, and it abbreviates CCTCC as, 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.Characteristics such as this method has simple and practical, and is with low cost are expected in industry, to apply.
Figure of description
For ease of understanding tobacco Arthrobacter molecular biological characteristics of the present invention, the spy combines accompanying drawing to further specify.
Fig. 1 is the 16SrDNA sequence chart of tobacco Arthrobacter.
Embodiment
To do further elaboration to the present invention through embodiment below, and its objective is to be better understanding content of the present invention.The example of therefore, being lifted does not limit protection scope of the present invention.
Embodiment 1
Slant culture: peptone 10g, yeast extract paste 5g, sodium-chlor 10g, agar 15g, zero(ppm) water 1000ml, pH7.0, with 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, the 0.5ml trace element, adding distil water 1000ml, adjust pH are 7.0, high pressure steam sterilization.It is 30ml that 250ml shakes bottled liquid measure, and inoculum size is 3v/v%, and culture temperature is 30 ℃, and shaking speed is 200rpm, shake-flask culture 36 hours, and the 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 dissolving constant volume 100ml.
Glycase enzyme activity (U) definition: under 45 ℃, pH7.0 condition, the required enzyme amount of hydrolysis 1mg starch in per 10 minutes.
Proteolytic enzyme enzyme activity (U) definition: under 37 ℃, pH7.0 condition, the PM caseinhydrolysate produces the required enzyme amount of 1 μ g tyrosine.
Embodiment 2
Slant culture is identical with embodiment 1.Shake the bottled 30ml of going into substratum at 250ml, it is formed as follows: nicotine 2g, steeping water 15g, yeast extract paste 5g, sodium-chlor 5g, K
2HPO
43H
2O13.3g, KH
2PO
44g, MgSO
47H
2O0.2g, the 0.5ml trace element, adding distil water 1000ml, adjust pH are 7.0, high pressure steam sterilization.It is 30ml that 250ml shakes bottled liquid measure, and inoculum size is 2v/v%, and culture temperature is 30 ℃, and shaking speed is 200rpm, shake-flask culture 48 hours, and the 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 glycase enzyme activity and proteolytic enzyme enzyme activity is identical with embodiment 1.
Embodiment 3
Get the fermented liquid 10ml among the embodiment 1, be diluted to 10 with sterile distilled water
9Individual bacterium/ml according to 1% of tobacco leaf weight, sprays application to bacterium liquid water cut and is among 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 descends 2.9%, protein contnt descends 5.6%, starch content reduces by 10.8%; The 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 among the embodiment 1, be diluted to 10 with sterile distilled water
9Individual bacterium/ml according to 1% of tobacco leaf weight, sprays application to bacterium liquid water cut and is among 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 descends 9.1%, protein contnt descends 12.2%, starch content reduces by 13.6%; The 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 among the embodiment 1, be diluted to 10 with sterile distilled water
9Individual bacterium/ml according to 1% of tobacco leaf weight, sprays application to bacterium liquid water cut and is among 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 descends 12%, protein contnt descends 18.5%, starch content reduces by 28.8%; The 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 among the embodiment 1, be diluted to 10 with sterile distilled water
9Individual bacterium/ml according to 2% of tobacco leaf weight, sprays application to bacterium liquid water cut and is among 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 descends 11.2%, protein contnt descends 21.6%, starch content reduces by 31.3%; The 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 among the embodiment 1, be diluted to 10 with sterile distilled water
9Individual bacterium/ml according to 2% of tobacco leaf weight, sprays application to bacterium liquid water cut and is among 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 descends 18.8%, protein contnt descends 25.3%, starch content reduces by 35.2%; The tobacco leaf pungency obviously reduces; Assorted gas reduces, and flue gas is soft, and cigarette is fragrant to be increased.
Claims (8)
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: said bacterial strain is a 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 SANMALT-S is negative, and wood sugar is negative, and ribose is negative; The yellow bacterium colony of smooth opaque, the neat in edge of when growth visible surface on substratum, optimum growth temperature is 25-30 ℃.
3. microorganism strains as claimed in claim 1 is characterized in that: said bacterial strain obtains through following approach:
At first from soil or tobacco leaf, isolate the microorganism strains of degrading nicotine;
Secondly select substratum to screen with milk the microorganism strains that obtains, acquisition can produce the bacterial strain of proteolytic enzyme;
Select screening of medium can produce diastatic bacterial strain with starch the bacterial strain that obtains then, acquisition is the nicotine degradation bacterium of degrade proteins and starch simultaneously, i.e. tobacco Arthrobacter Arthrobacter nicotianae GYC103.
4. like the arbitrary described microorganism strains of claim 1 to 3, it is characterized in that: the culturing step of said bacterial strain is:
A, slant culture: peptone 10g, yeast extract paste 5g, sodium-chlor 10g, agar 15g, zero(ppm) water 1000ml, pH7.0, with 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, the 0.5ml trace element, adding distil water 1000ml, adjust pH are 7.0, high pressure steam sterilization; Shaking bottled liquid measure is 10～30v/v%, and inoculum size is 2～10v/v%, and culture temperature is 20～40 ℃; Shaking speed is 100～200rpm, shake-flask culture 1～3 day, and the nicotine degradation rate reaches 85～100%; Proteinase activity reaches 18～60U/ml, and diastatic activity reaches 10～40U/ml.
5. microorganism strains as claimed in claim 4 is characterized in that: said 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 dissolving constant volume 100ml.
6. like the arbitrary described microorganism strains of claim 1 to 3, it is characterized in that: it can degrading nicotine, in process of growth, can produce glycase and proteolytic enzyme.
7. the application of a strain microorganism strains is characterized in that, said tobacco Arthrobacter Arthrobacter nicotianaeGYC103 is used for nicotine, starch and the protein of degrading tobacco; Said nicotine, starch and the protein that is used for degrading tobacco obtains through following steps:
A, the described tobacco Arthrobacter of claim 1 Arthrobacter nicotianae GYC103 is carried out fermentation culture, obtain fermented liquid and thalline;
B, fermented liquid or thalline water, saline water or the damping fluid of step a gained diluted, dilution back bacterial concentration reaches 10
6～10
12Individual bacterium/ml according to 1～5% of tobacco leaf weight, joins bacterium liquid water cut and is in 10～50% the tobacco and ferment, and leavening temperature is 10～50 ℃, and fermentation time was greater than 4 hours.
8. the application of microorganism strains as claimed in claim 6; It is characterized in that wherein said fermented liquid is 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 the phosphate buffered saline buffer any one in the steps A.
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| CN103373867A (en) * | 2013-06-24 | 2013-10-30 | 长沙碧野生态农业科技有限公司 | Technology for biochemical treatment and fertilizer use of tobacco invalidism body |
| CN103642722A (en) * | 2013-11-27 | 2014-03-19 | 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 |
| CN104164390A (en) * | 2014-07-09 | 2014-11-26 | 青岛蔚蓝天成生物科技有限公司 | Arthrobacter nicotianae and application thereof in aquaculture |
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| CN110195027A (en) * | 2019-03-08 | 2019-09-03 | 大连理工大学 | The preparation method and application of nicotianae ZL-1 and its compost low temperature fermentation inoculum |
| CN113773980A (en) * | 2021-07-31 | 2021-12-10 | 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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| 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 |
| CN103373867A (en) * | 2013-06-24 | 2013-10-30 | 长沙碧野生态农业科技有限公司 | Technology for biochemical treatment and fertilizer use of tobacco invalidism body |
| CN103642722A (en) * | 2013-11-27 | 2014-03-19 | 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 |
| CN104164390A (en) * | 2014-07-09 | 2014-11-26 | 青岛蔚蓝天成生物科技有限公司 | Arthrobacter nicotianae and application thereof in aquaculture |
| 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 |
| CN104388376A (en) * | 2014-11-07 | 2015-03-04 | 河南省烟草公司漯河市公司 | Nicotine-decomposing microorganism selective medium and preparation method thereof |
| CN109161569A (en) * | 2018-09-21 | 2019-01-08 | 郑州轻工业学院 | A kind of method of antimicrobial composition fermented tobacco extract production bacteria cellulose |
| 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 |
| CN110195027A (en) * | 2019-03-08 | 2019-09-03 | 大连理工大学 | The preparation method and application of nicotianae ZL-1 and its compost low temperature fermentation inoculum |
| 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 |
| CN113773980A (en) * | 2021-07-31 | 2021-12-10 | 广东中烟工业有限责任公司 | Enterobacter nicotianae NLB1 for degrading nicotine and application 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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