CN102719369A - Microbial strain and application thereof - Google Patents

Microbial strain and application thereof Download PDF

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Publication number
CN102719369A
CN102719369A CN2011101589749A CN201110158974A CN102719369A CN 102719369 A CN102719369 A CN 102719369A CN 2011101589749 A CN2011101589749 A CN 2011101589749A CN 201110158974 A CN201110158974 A CN 201110158974A CN 102719369 A CN102719369 A CN 102719369A
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nicotine
tobacco
microbacterium
culture
microorganism strains
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CN102719369B (en
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龙章德
黄泰松
韦建玉
白森
邹克兴
胡亚杰
蔡联合
金亚波
张纪利
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China Tobacco Guangxi Industrial Co Ltd
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China Tobacco Guangxi Industrial Co Ltd
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Abstract

The invention discloses a microbial strain and an application thereof, particularly relates to a tobacco nicotine degrading microbacterium GYC29 (Microbacterium sp.GYC29) with CCTCC of NO:M2010311 and an application of the microbacterium in tobacco. The strain disclosed herein can decompose and utilize nicotine in the growth process. According to the invention, by adding the ferment liquor of the strain or thallus with the additional amount of 1-5 % of the weight of the tobacco leaves in the tobacco with the water content of 10-50 % and fermenting for 6-72 h, the nicotine content of the tobacco leaves is reduced by 2-33%, the irritation of the tobacco leaves is obviously reduced, the miscellaneous gases are reduced, the smoke becomes pure and mild, and the sensory quality is obviously improved; the degradation of tobacco nicotine is realized by using microbes, the nicotine content of tobacco raw materials can be regulated properly, and the applicability of the tobacco leaves is raised.

Description

One strain microorganism strains and application thereof
Technical field
The present invention relates to a strain microorganism strains and an application thereof; Being specifically related to a kind of tobacco smoke alkaloid degradation bacteria---microbacterium GYC29 (Microbacterium sp.GYC29) CCTCC NO:M2010311 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.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.
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.Report like Deharyomyces nicotianae Micrococus nicotianae, Pseudomonas abroad; Alcaligenes paradox us; Arthrobacterglobif ormils, Enterobacter cloacae, Cunninghamella echinulata; Nicotianae plumbaginif olia, Arthrobacter oxidans etc.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).Utilize mikrobe to handle tobacco leaf, the nicotine in the tobacco leaf of not only having degraded improves the quality of tobacco leaf, also reduces the pollution to environment, improves the utilization ratio of tobacco leaf resource, has high economic benefit and social benefit.Therefore, the mikrobe that screening can efficiently degrading nicotine is used for handling the nicotine of tobacco tobacco leaf, is with a wide range of applications.In the nicotine degradation bacterium of having reported, Shang Weiyou utilizes the report of microbacterium degrading nicotine.
Summary of the invention
It is microbacterium GYC29 (Microbacterium sp.GYC29) that main purpose of the present invention is to provide a kind of microorganism strains that can efficiently degrading nicotine; Preserving number is CCTCC NO:M2010311, and corresponding culture condition also discloses a kind of method of utilizing the nicotine 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 microbacterium Microbacterium sp.GYC29, and preserving number is CCTCC NO:M2010311; Bacterium colony is yellow, and the smooth surface opaque border is neat; Gram-positive, optimum growth temperature are 25-30 ℃.
Said bacterial strain obtains through following approach:
Tobacco-growing soil is got 10g respectively to join in the triangular flask that contains the 200mL sterilized water; In 30 ℃; The shaking table of 200rpm shakes 30min, filters, and gets the 1mL bacterial suspension and is forwarded in another triangular flask that contains the 200mL enrichment medium; Enrichment culture 3d gets the 1mL culture then and is forwarded to another and contains in the triangular flask that the 200mL nicotinic density is 0.2% nicotine substratum and cultivate 3d.Culture dilution is applied to nicotine selects on the substratum, through separation and purification and tolerance nicotine ability test, obtain in 0.8% nicotinic density nutrient solution can vigorous growth the degrading nicotine bacterial strain, i.e. microbacterium Microbacterium sp.GYC29.
Said enrichment medium is: peptone 10g, yeast extract 5.0g, NaCl 10g, 1g K 2HPO 4, water 1000ml, pH7.0.
Said nicotine substratum is: 0.2w/v% nicotine, 13.3g K 2HPO 43H 2O, 4g KH 2PO 4, 0.2g MgSO 47H 2O, 0.05ml trace element, water 1000ml, pH7.0.
Said trace element is: 0.04g CaCl 2, 0.07g CuSO 4, 0.008g MnSO 4H 2O, 0.004g FeSO 47H 2O, 0.1g Na 2MoO 42H 2O adds 0.1molL -1HCl dissolving constant volume 100mL.
Said nicotine selects substratum to be: nicotine substratum+1.5w/v% agar powder.
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 2O 13.3g, KH 2PO 44g, MgSO 47H 2O 0.2g, 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 ℃, and shaking speed is 100～200rpm, shake-flask culture 1～3 day, and the nicotine degradation rate reaches 85～100%.
Said trace element is: CaCl 20.04g, CuSO 40.07g, MnSO 4H 2O 0.008g, FeSO 47H 2O 0.004g, Na 2MoO 42H 2O 0.1g adds 0.1molL -1HCl dissolving constant volume 100ml.Said microbacterium Microbacterium sp.GYC29 is used for the nicotine of degrading tobacco; The said nicotine that is used for degrading tobacco obtains through following steps:
A, the described microbacterium Microbacterium of claim 1 sp.GYC29 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 not less than 4 hours.
Wherein said damping fluid 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 among the step B.
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, nicotine content of tobacco leaves is reduced, pungency obviously reduces, smoke alcohol with, tobacco leaf usability obviously improves.
Said microbacterium Microbacterium sp.GYC29 is deposited in Chinese typical culture collection center on November 24th, 2010, and it abbreviates CCTCC as, and deposit number is CCTCC NO:M2010311.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, nicotine content of tobacco leaves reduces, and pungency obviously reduces, smoke alcohol with, 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 the molecular biological characteristics of microbacterium GYC29 of the present invention, the spy combines accompanying drawing to further specify.
Fig. 1 is the 16S rDNA sequence chart of microbacterium GYC29.
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 2O 13.3g, KH 2PO 44g, MgSO 47H 2O 0.2g, 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 20 ℃, and shaking speed is 200rpm, shake-flask culture 24 hours, and the nicotine degradation rate reaches 100%.Trace element: CaCl 20.04g, CuSO 40.07g, MnSO 4H 2O 0.008g, FeSO 47H 2O 0.004g, Na 2MoO 42H 2O 0.1g adds 0.1molL -1HCl dissolving constant volume 100ml.
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 4g, steeping water 15g, yeast extract paste 5g, sodium-chlor 5g, K 2HPO 43H 2O 13.3g, KH 2PO 44g, MgSO 47H 2O 0.2g, 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 85%.
Embodiment 3
Slant culture is identical with embodiment 1.Shake the bottled 30ml of going into substratum at 250ml, it is formed as follows: nicotine 8g, steeping water 15g, yeast extract paste 5g, sodium-chlor 5g, K 2HPO 43H 2O 13.3g, KH 2PO 44g, MgSO 47H 2O 0.2g, 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 40 ℃, and shaking speed is 200rpm, shake-flask culture 72 hours, and the nicotine degradation rate reaches 62%.
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 16%; 35 ℃, the condition bottom fermentation of relative humidity 65% 24 hours; Can make nicotine content of tobacco leaves descend 8.6%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.
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 16%; 35 ℃, the condition bottom fermentation of relative humidity 65% 36 hours; Can make nicotine content of tobacco leaves descend 12.9%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.
Embodiment 6
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 16%; 35 ℃, the condition bottom fermentation of relative humidity 65% 48 hours; Can make nicotine content of tobacco leaves descend 20%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.
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 16%; 35 ℃, the condition bottom fermentation of relative humidity 75% 60 hours; Can make nicotine content of tobacco leaves descend 26.2%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.
Embodiment 8
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 16%; 35 ℃, the condition bottom fermentation of relative humidity 65% 72 hours; Can make nicotine content of tobacco leaves descend 32.8%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.
Embodiment 9
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 10%; 50 ℃, the condition bottom fermentation of relative humidity 65% 24 hours; Can make nicotine content of tobacco leaves descend 8.6%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.
Embodiment 10
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 25%; 35 ℃, the condition bottom fermentation of relative humidity 65% 36 hours; Can make nicotine content of tobacco leaves descend 12.9%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.
Embodiment 11
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 30%; 35 ℃, the condition bottom fermentation of relative humidity 65% 48 hours; Can make nicotine content of tobacco leaves descend 20%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.
Embodiment 12
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 40%; 35 ℃, the condition bottom fermentation of relative humidity 75% 60 hours; Can make nicotine content of tobacco leaves descend 26.2%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.
Embodiment 13
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 50%; 35 ℃, the condition bottom fermentation of relative humidity 65% 4 hours; Can make nicotine content of tobacco leaves descend 32.8%, the tobacco leaf pungency obviously reduces, and assorted gas reduces; It is pure and mild that flue gas becomes, and quality of tobacco improves.

Claims (6)

1. microorganism strains is characterized in that: it is microbacterium Microbacterium sp.GYC29, and preserving number is CCTCC NO:M2010311; Bacterium colony is yellow, and the smooth surface opaque border is neat; Gram-positive, optimum growth temperature are 25-30 ℃.
2. 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; Select substratum to screen with nicotine the microorganism strains that obtains then, through separation and purification and tolerance nicotine ability test, the bacterial strain that acquisition can efficiently degrading nicotine, i.e. microbacterium Microbacterium sp.GYC29.
3. according to claim 1 or claim 2 microorganism strains, 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 2O 13.3g, KH 2PO 44g, MgSO 47H 2O 0.2g, 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 ℃, and shaking speed is 100～200rpm, shake-flask culture 1～3 day, and the nicotine degradation rate reaches 85～100%.
4. microorganism strains as claimed in claim 3 is characterized in that: said trace element is: CaCl 20.04g, CuSO 40.07g, MnSO 4H 2O 0.008g, FeSO 47H 2O 0.004g, Na 2MoO 42H 2O 0.1g adds 0.1molL -1HCl dissolving constant volume 100ml.
5. the application of the described strain microorganism strains of claim 1 is characterized in that, said microbacterium Microbacterium sp.GYC29 is used for the nicotine of degrading tobacco; The said nicotine that is used for degrading tobacco obtains through following steps:
A, the described microbacterium Microbacterium of claim 1 sp.GYC29 is carried out fermentation culture, obtain fermented liquid and thalline;
B, fermented liquid or thalline water, saline water or the damping fluid of steps 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 not less than 4 hours.
6. the application of microorganism strains as claimed in claim 5; It is characterized in that wherein said damping fluid 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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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104371948A (en) * 2014-07-02 2015-02-25 中国中化股份有限公司 Microbacterium sp. strain and application thereof
CN113755363A (en) * 2021-07-26 2021-12-07 南阳师范学院 Preparation and application of Mixta calida bacteria for degrading nicotine
CN113980839A (en) * 2021-10-14 2022-01-28 华南农业大学 Delftia sp NLG11 for degrading tobacco nicotine and application thereof
CN114891694A (en) * 2022-06-10 2022-08-12 四川中烟工业有限责任公司 Acinetobacter indiani and application thereof

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104371948A (en) * 2014-07-02 2015-02-25 中国中化股份有限公司 Microbacterium sp. strain and application thereof
CN104371948B (en) * 2014-07-02 2017-02-01 中国中化股份有限公司 Microbacterium sp. strain and application thereof
CN113755363A (en) * 2021-07-26 2021-12-07 南阳师范学院 Preparation and application of Mixta calida bacteria for degrading nicotine
CN113980839A (en) * 2021-10-14 2022-01-28 华南农业大学 Delftia sp NLG11 for degrading tobacco nicotine and application thereof
CN113980839B (en) * 2021-10-14 2023-04-18 华南农业大学 Delftia sp NLG11 for degrading tobacco nicotine and application thereof
CN114891694A (en) * 2022-06-10 2022-08-12 四川中烟工业有限责任公司 Acinetobacter indiani and application thereof
CN114891694B (en) * 2022-06-10 2024-03-12 四川中烟工业有限责任公司 Acinetobacter indicum and application thereof

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