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
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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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