CN106350467B - Reduce the bacterial strain and screening technique, cultural method and application of nicotine in tobacco leaf - Google Patents
Reduce the bacterial strain and screening technique, cultural method and application of nicotine in tobacco leaf Download PDFInfo
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- CN106350467B CN106350467B CN201610786460.0A CN201610786460A CN106350467B CN 106350467 B CN106350467 B CN 106350467B CN 201610786460 A CN201610786460 A CN 201610786460A CN 106350467 B CN106350467 B CN 106350467B
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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
- C12R2001/07—Bacillus
- C12R2001/085—Bacillus cereus
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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
- 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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- 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
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Abstract
The invention discloses a kind of bacterial strains of nicotine in reduction tobacco leaf, and the bacterial strain is Bacillus cercus, is obtained by following approach: taking 9~10g of pedotheque in 90mL enriched medium, then cultivate 40~50h at 28~32 DEG C of temperature and obtain enrichment culture liquid；Enrichment culture 1~1.2mL of liquid is taken, the culture dish equipped with the isolation medium for being cooled to 48~52 DEG C after fusing is put into, solidification, which is placed in 28~32 DEG C of temperature of constant incubator, cultivates 6~8 days；Then it selects in the bacterium colony access domestication culture medium of growth, 90~110h is cultivated under conditions of 28~32 DEG C of Yu Wendu, 100~150rpm of revolving speed；Then culture gradient dilution is coated on isolation medium plate, obtains single colonie, isolated and purified by scribing line.Using high, the sugared alkali of strain fermentation processing nicotine content than the tobacco leaf of numerous imbalances, nicotine content 20~40% can be reduced.
Description
Technical field
The present invention relates to a kind of microbial strains, and in particular to one kind can reduce the Bacillus cercus of nicotine in tobacco leaf
The application of (Bacillus cereus) and its screening technique, cultural method and the bacterial strain in tobacco leaf.
Background technique
Tobacco leaf is the raw material of production of cigarettes, and the quality of quality of tobacco is directly related to the height of cigarette quality.The change of tobacco leaf
Learning conventional ingredient is mainly: total alkaloid, Water-soluble Total Sugar, reduced sugar, total nitrogen, chlorine and potassium also contain protein, starch, fibre
The macromolecular compounds such as element, pectin are tieed up, these ingredients are in the content in tobacco leaf and the proportionate relationship between certain ingredients, to cigarette
Leaf quality has important influence.Nicotine also known as nicotine (Nicotine), are the main components in nicotiana alkaloids.In flue-cured tobacco
The content of nicotine is advisable generally between 1.5%~3.5% with 2.5% or so.Nicotine content is too low, and strength is too small, lacks full
Foot sense, nicotine content is excessively high, and strength is too big, can cause to pierce pungent sense of choking.China part inventory's tobacco leaf (especially upper tobacco leaf)
It is excessively high that there are nicotine contents, and sugared alkali is than numerous imbalances, the problem of the quality and poor availability of tobacco leaf.
Currently, there are mainly three types of approach for the method for reduction nicotine content in tobacco leaf: 1. from agriculturals such as heredity, ecology, plantations
Plantation aspect carries out nicotine content regulation；2. using the processing such as hot water rinsing, organic solvent extraction, gas extracting, steam distillation
Tobacco leaf, chemically angle removes nicotine in tobacco leaf；3. handling tobacco leaf using microorganism or biotechnology of enzymes, nicotine in tobacco leaf is reduced
Content improves the availability of tobacco leaf.It, can only be using two kinds of approach below for the mature tobacco leaf harvested.Wherein, it uses
Though chemical method can remove nicotine, the loss of some fragrance matters in tobacco leaf will lead to simultaneously, and pass through microorganism or enzyme
Tobacco leaf is handled, since enzyme has high specificity, thus can preferably avoid the problem of tobacco leaf fragrance component damages.
Summary of the invention
It is excessively high for certain inventory's nicotine content in tobacco leaf, sugared alkali than numerous imbalances, the quality of tobacco leaf and poor availability
Problem, the present invention provide a kind of Bacillus cercus (Bacillus cereus) that can reduce nicotine in tobacco leaf；
Another object of the present invention is to provide the screening sides of the Bacillus cercus (Bacillus cereus)
Method；
There is provided the cultural methods of Bacillus cercus (Bacillus cereus) for a further object of the present invention；
There is provided Bacillus cercus (Bacillus cereus) answering in tobacco leaf for a further object of the present invention
With.
In order to reach above-mentioned technical effect, the present invention takes following technical scheme:
The bacterial strain of nicotine in a kind of reduction tobacco leaf, the bacterial strain are Bacillus cercus (Bacillus cereus), should
Bacterial strain is preserved in China General Microbiological culture presevation administrative center, deposit number are as follows: CGMCC 10338.
The present invention provides the culture of the Bacillus cercus and micro-morphology are as follows:
Bacterium colony is big on nutrient agar plate, coarse, and irregularly, there are whiplike branch, micro white in edge, and surface has characteristic
Speckle；
Vigorous, coarse, opaque, pale is grown on nutrient agar inclined-plane, is extended, edge is irregular, there is whiplike branch；
Cell is rod-shaped, 3.0-4.8 × 1.0-1.1um, end side, at short chain.
The present invention also very provides the screening technique for reducing the bacterial strain of nicotine in tobacco leaf, comprising the following steps:
Step A: taking 9~10g of pedotheque in 90mL enriched medium, then at 28~32 DEG C of temperature culture 40~
50h obtains enrichment culture liquid；
Step B: taking enrichment culture 1~1.2mL of liquid, is put into equipped with the isolation medium for being cooled to 48~52 DEG C after fusing
Culture dish, solidification, which is placed in 28~32 DEG C of temperature of constant incubator, cultivates 6~8 days；Then the bacterium colony access of growth is selected
It tames in culture medium, 90~110h is cultivated under conditions of 28~32 DEG C of Yu Wendu, 100~150rpm of revolving speed；Then by culture
Gradient dilution is coated on isolation medium plate, obtains single colonie, isolates and purifies to obtain the wax-like gemma bar by crossing
Bacterium (Bacillus cereus).
In above-mentioned screening technique, the enriched medium includes the component of following weight: peptone, 5.0g；
NaCl, 1.0g；K2HPO4, 1.0g；Agar, 1.5g；The pH of enriched medium is 7.0,121 DEG C of sterilizing 30min, is added after cooling
Nicotine 2.0g.
In above-mentioned screening technique, the isolation medium includes the component of following weight or volume: K2HPO4,
1.0g；MgSO4, 0.5g；The MnSO that mass fraction is 0.1%4, 1mL；The FeSO that mass fraction is 0.1%4, 3mL；Agar,
1.5g；Nicotine 4.0g is added after cooling in 6.0~7.0,121 DEG C of sterilizing 30min of pH of isolation medium.
In above-mentioned screening technique, the domestication culture medium includes the component of following weight or volume: K2HPO4,
1.0g；MgSO4, 0.5g；The MnSO that mass fraction is 0.1%4, 1mL；The FeSO that mass fraction is 0.1%4, 3mL；Domestication culture
Nicotine 2000mg is added after cooling in 6.0~7.0,121 DEG C of sterilizing 30min of pH of base.
The present invention also provides the cultural methods for reducing the bacterial strain of nicotine in tobacco leaf, comprising the following steps:
Step a: inclined-plane culture: the Bacillus cercus of freezen protective is inoculated on slant medium, and Yu Wendu 28~
Stationary culture 1~2 day at 32 DEG C；
Step b: shaking flask Liquid Culture: cultivating resulting strain for step a and be inoculated on fluid nutrient medium, and Yu Wendu 28~
32 DEG C, shaking flask culture 18 under conditions of 140~160r/min of revolving speed~for 24 hours, culture to bacterium number is reached up to 1010More than, obtain seed
Liquid.
In above-mentioned cultural method, the slant medium or fluid nutrient medium include the group of following weight or volume
Point: peptone 10g, beef extract 3g, sodium chloride 5g, agar 15g, distilled water 1000mL, pH value 7.0~7.2.
The present invention also provides the applications of the bacterial strain for reducing nicotine in tobacco leaf nicotine in reducing tobacco leaf, by seed
Liquid is added in tobacco leaf according to the 5~30% of tobacco leaf weight, is then fermented 1~6 day at 20~45 DEG C of temperature.
In the applications described above, it is preferred that by seed liquor according to the 20~30% of tobacco leaf weight, it is added in tobacco leaf,
Then it ferments 1~6 day at 20~45 DEG C of temperature.
The present invention provides one plant of Bacillus cercus (Bacillus cereus), the strain growth reproductive capacity is strong, mycelia
It grows vigorous.Tobacco leaf using high, the sugared alkali of strain fermentation processing nicotine content than numerous imbalances, can reduce nicotine content 20~
40%, there is preferable stability and reproducibility, tobacco leaf strength is moderate after fermentation, chokes and pierces peppery sense reduction, and harmony improves, sense organ
Quality is obviously improved.
Detailed description of the invention
Fig. 1 is the colonial morphology figure of Bacillus cercus (Bacillus cereus)；
Fig. 2 is the cellular morphology figure of Bacillus cercus (Bacillus cereus)；
The preservation of Bacillus cercus (Bacillus cereus) illustrates:
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC；Preservation
Location: the institute 3 of Chaoyang District Beijing North Star West Road 1；
Deposit number: CGMCC No.10338；
The preservation time: on January 12nd, 2015.
Specific embodiment
Below with reference to the embodiment of the present invention, the invention will be further elaborated.
Embodiment 1: separation, the identification of Bacillus cercus
Taking pedotheque (Sichuan Liangshan District ginger state vega soil), 10g is in 90mL enriched medium, 30 DEG C of stationary cultures
48h.Enrichment culture liquid 1mL is taken, is put into equipped with being uniformly mixed in the culture dish for being cooled to 50 DEG C of isolation mediums after fusing, solidifies
After be put in 30 DEG C of constant incubator cultures 7 days.In the bacterium colony access domestication culture medium of picking growth, 30 DEG C, 120rpm culture
Culture energy gradient dilution is coated on isolation medium plate, obtains single colonie by 96h, isolates and purifies wax-like by scribing line
Bacillus strain (Bacillus cereus).
The enriched medium are as follows: peptone, 5.0g；NaCl, 1.0g；K2HPO4, 1.0g；Agar, 1.5g；Enrichment culture
The pH of base is 7.0,121 DEG C of sterilizing 30min, and nicotine 2.0g is added after cooling.
The isolation medium are as follows: K2HPO4, 1.0g；MgSO4, 0.5g；The MnSO that mass fraction is 0.1%4, 1mL；Matter
Measure the FeSO that score is 0.1%4, 3mL；Agar, 1.5g；6.0~7.0,121 DEG C of the pH sterilizing 30min of isolation medium is cold
But nicotine 4.0g is added afterwards.
The domestication culture medium are as follows: K2HPO4, 1.0g；MgSO4, 0.5g；The MnSO that mass fraction is 0.1%4, 1mL；Matter
Measure the FeSO that score is 0.1%4, 3mL；6.0~7.0,121 DEG C of sterilizing 30min of pH of culture medium are tamed, cigarette is added after cooling
Alkali 2000mg.
The culture of said waxy bacillus and micro-morphology are described as follows:
Bacterium colony is big on nutrient agar plate, coarse, and irregularly, there are whiplike branch, micro white in edge, and surface has characteristic
Speckle.
Vigorous, coarse, opaque, pale is grown on nutrient agar inclined-plane, is extended, edge is irregular, there is whiplike branch,
Cell is rod-shaped, 3.0-4.8 × 1.0-1.1um, end side, at short chain.
16S rRNA gene order and danj gene order identified, that Bacillus cercus has.
Embodiment 2: the preparation of Bacillus cercus seed liquor
The Bacillus cercus bacterium colony that embodiment 1 obtains is expanded culture and produced, Bacillus cercus kind is obtained
Sub- liquid.
A, inclined-plane culture: peptone 10g, beef extract 3g, sodium chloride 5g, agar 15g, distilled water 1000mL, pH value 7.0~
7.2, the Bacillus cercus strain (Bacillus cereus) of freezen protective is inoculated on slant medium, 30 DEG C of constant temperature are quiet
Only cultivate 1-2 days.
B, shaking flask Liquid Culture: peptone 10g, beef extract 3g, sodium chloride 5g, agar 15g, distilled water 1000mL, pH value
7.0~7.2, high pressure steam sterilization, by step A thallus be inoculated into medium liquid, cultivation temperature: 30 DEG C, shaking table turn
Fast 140-160r/min, shaking flask culture 18-24h, culture to bacterium number is up to 1010More than, obtain seed liquor.
Embodiment 3:
The Bacillus cercus seed liquor that embodiment 2 is obtained is added to water content 15% according to the 30% of tobacco leaf weight
Tobacco leaf in, ferment 6 days under the conditions of 25 DEG C, compared with non-fermentation process blank tobacco leaf, in fermenting tobacco leaf nicotine content reduce
24.9%, Analyses Methods for Sensory Evaluation Results shows: fermenting tobacco leaf strength is substantially reduced, and irritation significantly reduces, flue gas is more pure and mild.
Embodiment 4:
The Bacillus cercus seed liquor that embodiment 2 is obtained is added to water content 50% according to the 5% of tobacco leaf weight
It in tobacco leaf, ferments 3 days, compared with non-fermentation process blank tobacco leaf, nicotine content reduces by 29.7% in fermenting tobacco leaf, sensory evaluation
The result shows that: fermenting tobacco leaf strength is substantially reduced, and irritation significantly reduces, flue gas is more pure and mild.
Embodiment 5:
The Bacillus cercus seed liquor that embodiment 2 is obtained is added to water content 35% according to the 20% of tobacco leaf weight
Tobacco leaf in, ferment 4 days, compared with non-fermentation process blank tobacco leaf, in fermenting tobacco leaf nicotine content reduce by 38.1%, sense organ is commented
Valence the result shows that: fermenting tobacco leaf strength is substantially reduced, and irritation significantly reduces, flue gas is more pure and mild.
Although reference be made herein to invention has been described for explanatory embodiment of the invention, and above-described embodiment is only this hair
Bright preferable embodiment, embodiment of the present invention are not limited by the above embodiments, it should be appreciated that those skilled in the art
Member can be designed that a lot of other modification and implementations, these modifications and implementations will fall in principle disclosed in the present application
Within scope and spirit.
Claims (4)
1. a kind of bacterial strain for reducing nicotine in tobacco leaf, it is characterised in that the bacterial strain is Bacillus cercus (Bacillus
Cereus), which is preserved in China General Microbiological culture presevation administrative center, deposit number are as follows: CGMCC 10338.
2. the bacterial strain according to claim 1 for reducing nicotine in tobacco leaf, it is characterised in that the Bacillus cercus
Culture and micro-morphology are as follows:
Bacterium colony is big on nutrient agar plate, coarse, and irregularly, there are whiplike branch, micro white in edge, and surface has characteristic spot
Line；
Vigorous, coarse, opaque, pale is grown on nutrient agar inclined-plane, is extended, edge is irregular, there is whiplike branch；Cell
It is rod-shaped, 3.0-4.8 × 1.0-1.1um, end side, at short chain.
3. the application of the bacterial strain for reducing nicotine in tobacco leaf nicotine in reducing tobacco leaf described in claim 1, it is characterised in that will
Seed liquor is added in tobacco leaf according to the 5~30% of tobacco leaf weight, is then fermented 1~6 day at 20~45 DEG C of temperature；
The seed liquor is made of following steps:
Step a: inclined-plane culture: the Bacillus cercus of freezen protective is inoculated on slant medium, and 28~32 DEG C of Yu Wendu
Lower stationary culture 1~2 day；
Step b: shaking flask Liquid Culture: cultivating resulting strain for step a and be inoculated on fluid nutrient medium, Yu Wendu 28~32
DEG C, shaking flask culture 18 under conditions of 140~160r/min of revolving speed~for 24 hours, culture to bacterium number reaches 1010More than, obtain seed liquor.
4. the application of bacterial strain according to claim 3 nicotine in reducing tobacco leaf, it is characterised in that by seed liquor according to cigarette
The 20~30% of leaf weight, are added in tobacco leaf, then ferment 1~6 day at 20~45 DEG C of temperature.
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| EP3313173A1 (en) | 2015-06-26 | 2018-05-02 | Altria Client Services LLC | Compositions and methods for producing tobacco plants and products having altered alkaloid levels |
| CN113215062A (en) * | 2021-06-15 | 2021-08-06 | 四川中烟工业有限责任公司 | Bacillus amyloliquefaciens, and acquisition method and application thereof |
| CN115191636A (en) * | 2022-06-03 | 2022-10-18 | 云南瑞升烟草技术（集团）有限公司 | Reconstituted tobacco with high sugar-alkali ratio and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102168051A (en) * | 2011-01-25 | 2011-08-31 | 浙江工业大学 | Bacillus cereus with higher resistance to jinggangmycin aqua and application |
| CN102266118A (en) * | 2011-06-03 | 2011-12-07 | 川渝中烟工业公司 | Preparation method of tobacco leaves of cigars |
| CN103082402A (en) * | 2013-02-06 | 2013-05-08 | 川渝中烟工业有限责任公司 | Wettable powder for cigar tobacco fermentation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168051A (en) * | 2011-01-25 | 2011-08-31 | 浙江工业大学 | Bacillus cereus with higher resistance to jinggangmycin aqua and application |
| CN102266118A (en) * | 2011-06-03 | 2011-12-07 | 川渝中烟工业公司 | Preparation method of tobacco leaves of cigars |
| CN103082402A (en) * | 2013-02-06 | 2013-05-08 | 川渝中烟工业有限责任公司 | Wettable powder for cigar tobacco fermentation |
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