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Bacillus pumilus, method for acquiring strain and application of strain in orientated degradation of nitrosoamine specifically in tobacco

A technology for obtaining Bacillus pumilus, which is applied in the field of Bacillus pumilus, can solve the problem that there are not many ways to degrade TSNAs, and achieve the effects of good stress resistance, high activity, and easy separation

Active Publication Date: 2014-03-26
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The formation of TSNAs can be regulated by changing these influencing factors, but there are not many methods for TSNAs degradation

Method used

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  • Bacillus pumilus, method for acquiring strain and application of strain in orientated degradation of nitrosoamine specifically in tobacco
  • Bacillus pumilus, method for acquiring strain and application of strain in orientated degradation of nitrosoamine specifically in tobacco
  • Bacillus pumilus, method for acquiring strain and application of strain in orientated degradation of nitrosoamine specifically in tobacco

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] ——The acquisition and identification of Bacillus pumilus 05-5402

[0076] (1) Acquisition of Bacillus pumilus 05-5402

[0077] A. Separation: Leaf samples of flue-cured tobacco K326 were collected from Yuxi City, Yunnan Province. 1 g of the tobacco leaf sample was subjected to tissue grinding, 100 ml of sterile water was added, and the extract was shaken and extracted at room temperature at 150 rpm for 30 min to obtain an extraction suspension. The extracted suspension is separated from the strains that can grow on the separation medium by the dilution plate method;

[0078] The culture condition is 28°C and the time is 48h;

[0079] The components of the separation medium are calculated in g / L: agar 15.0, K 2 HPO 4 1.6、KH 2 PO 4 0.4, NaCl0.1, MgSO 4 ·7H 2 O0.2, CaCl 2 0.05, MnSO 4 ·H2O0.002、CuSO 4 ·5H 2 O0.0001, ZnSO 4 ·7H 2 O0.0002, NaMoO 4 2H 2 O0.0002, nicotine 1.0, pH7.0.

[0080] B, screening: transfer the isolated bacterial strain to the screenin...

Embodiment 2

[0098] ——Degradation experiment of Bacillus pumilus 05-5402 on TSNAs in tobacco extract

[0099] Experiment method: mix according to the ratio of burley tobacco powder: distilled water = 1:10, ultrasonically extract for 30 minutes and then filter, add yeast extract (1.5g / L) to the filtrate, adjust the pH to 7.2-7.4. Pack 100ml in a 300ml Erlenmeyer flask Sterilize, pick a circle of screened strain 05-5402 to inoculate, shake and culture at 150rpm for 48h. Burley tobacco extract without inoculation was used as control (CK). Bacteria solution was centrifuged at room temperature at a speed of 8000rpm for 10min, and the filtrate was filtered through a 0.22 μm water phase membrane, and the filtrate was taken for UPLC-MS / MS analysis (Fan Duoqing, etc., Chinese Journal of Tobacco, 2012, 18(6): 10-16 ).

[0100] Experimental results: From the data in Table 1, it can be seen that the strain 05-5402 has a good effect on the degradation of TSNAs in the extract of Burley tobacco. After...

Embodiment 3

[0104] ——Degradation experiment of Bacillus pumilus 05-5402 on TSNAs in cured tobacco leaves

[0105] experimental method:

[0106] a, fermentation: first inoculate Bacillus pumilus (Bacillus pumilus) 05-5402 on the slant medium to obtain fermented seeds;

[0107] The culture conditions are: 26°C, time 24h;

[0108] The components of the slant medium are calculated in g / L: peptone 10.0, beef extract 3.0, NaCl 5.0, agar 17.0, pH 7.2;

[0109] Then the fermented seeds are inoculated into the seed culture solution, the inoculum amount is 4v / v%, the liquid volume of the shaker bottle is 30v / v%, and the shaker bottle rotation speed is 150rpm to obtain the fermentation inoculum;

[0110] The culture conditions are: 28°C, time 36h;

[0111] The components of the seed culture solution in g / L were: tryptone 10.0, yeast extract 5.0, NaCl 5.0.

[0112] The effective number of viable bacteria of the obtained fermented bacterial agent is 1.2×10 9 pieces / ml.

[0113] b. Inoculation: ...

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Abstract

The invention discloses bacillus pumilus, a method for acquiring the strain, and application of the strain in orientated degradation on nitrosoamine specifically in tobacco, and belongs to the technical field of microorganism application. The bacillus pumilus 05-5402 is preserved in the Common Microorganism Center of the Chinese Microorganism Strain Preservation Administration Committee, and the preservation number is CGMCC No. 7418. The method for acquiring the strain comprises procedures of separation, screening and purification, namely, firstly separating a strain which can degrade nicotine from tobacco plant or soil, further screening by using a culture medium which takes NNK (Tobacco-Specific Nitrosamine) as a sole carbon source and nitrogen source, and finally purifying on a culture medium plate so as to obtain the bacillus pumilus. The application of the bacillus pumilus comprises procedures of fermentation, inoculation and degradation, namely, firstly culturing the strain for 48-72 hours at 25-30 DEG C so as to obtain a zymophyte agent, spraying the zymophyte agent according to a percentage of 3-5% of the weight of the tobacco in the tobacco leaf conditioning or slicing process, and keeping the degradation time of 4-7 days. Through the adoption of the strain, high-efficient orientated degradation on nitrosoamine specifically in tobacco is achieved, and the strain is good in stress resistance, simple and convenient to obtain and convenient to use, and has high popularization and application values.

Description

technical field [0001] The invention belongs to the technical field of microbial application, and in particular relates to a bacillus pumilus strain capable of realizing the directional degradation of tobacco-specific nitrosamines, an acquisition method thereof, and an application in the directional degradation of tobacco-specific nitrosamines. Background technique [0002] TSNAs are tobacco-specific N-nitroso compounds, which are important harmful components in tobacco leaves and have a serious impact on the health of smokers. NNN, NNK, NAB, and NAT are the main TSNAs in tobacco and smoke, especially NNN and NNK are strong carcinogens in animals, which are extremely harmful. There are many factors that affect the content of TSNAs in tobacco, including tobacco type, tobacco tissue, cultivation method, preparation method, microbial community, nitrogen fertilizer application rate, nitrate reductase and nitrosase activity, etc. The formation of TSNAs can be regulated by changi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02A24B15/20C12R1/07
Inventor 夏振远雷丽萍汪安云吴玉萍莫笑晗马雁军周俊
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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