A strain of Bacillus Velezii DX-32 capable of efficiently degrading nitrite and TSNAs and its application

Abstract

The present invention relates to a strain of Bacillus velezensis DX-32 that can efficiently degrade nitrite and TSNAs and its application, and belongs to the field of microorganisms. The present invention separates and screens Bacillus velezensis DX-32 from cigar tobacco leaves, which has been preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 2022908. The Bacillus velezensis DX-32 can not only efficiently degrade nitrite, but also significantly reduce tobacco-specific nitrosamines (TSNAs), which is of great significance in improving tobacco safety, and also provides a valuable microbial resource for tobacco agricultural harm reduction.

Description

Technical Field

[0001] The invention relates to Bacillus velezensis DX-32 with dual functions of degrading nitrite and TSNAs and application thereof, belonging to the field of microorganisms. Background Art

[0002] Tobacco-Specific Nitrosamines (TSNAs) are a group of carcinogens found only in tobacco, its tobacco products and smoke, and are the main harmful components in tobacco. They are the products of nitrosation reactions between nitrogen-containing compounds (such as nicotine, demethylnicotine, anabasine and anatabine, etc.) and nitrite derivatives (such as N02, N203, and N204, etc.) under acidic conditions. The TSNAs identified so far are mainly composed of four substances: 4-(N-nitrosomethylamin-l-(3-pyridyl)-1-butanone (NNK), N-nitrosonornieotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanabine (NAT). Among them, NNN and NNK are the main carcinogens (Gupt a, PC; Murti, PR; Bhonsle, RB Epidemiology of cancer by tobacco products and the significance of tsna. Crit. Rev. Toxicol. 1996, 26, 183-198.). NNN and NNK have been listed as harmful ingredients by the International Agency for Research on Cancer (IARC) and the U.S. Food and Drug Administration (FDA), therefore, reducing the content of TSNAs in tobacco and tobacco products is crucial to reduce their harmful effects.

[0003] At present, TSNAs are degraded by endophytes that can degrade nitrate and nitrite, but nitrite is produced while degrading nitrate. This may be because nitrate is reduced to nitrite. The strains with strong nitrate degradation ability have higher nitrate reductase activity in their bodies, which leads to its conversion to nitrite. Yang Huanwen et al. confirmed that during the drying and aging of burley tobacco, the nitrate reduction of microorganisms increased the nitrite content in tobacco leaves, leading to the accumulation of TSNAs (Yang Huanwen, et al. (1998) Formation, accumulation and influencing factors of tobacco-specific N-nitrosamines. Tobacco Science and Technology, 31-34.). One of the current ways to reduce TSNAs is to use strains that can efficiently degrade tobacco nitrite but cannot degrade nitrate. Giovanni (Giovanni V, Michele DG, Elisabetta C. (2007) Nitrite metabolism in Debaryomyces hansenii TOB-Y7, a yeast strain involved in tobacco fermentation. Appl Microb Biotecl. 75: 633-645.) isolated and screened a yeast strain Debaryomyces hansenii TOB-Y7 from the surface of fermented tobacco leaves. This yeast strain lacks nitrate reductase and cannot utilize nitrate, but has the ability to efficiently degrade nitrite. Compared with the traditional fermentation method, it can significantly reduce nitrite and TSNAs when used in tobacco fermentation. Therefore, the present invention reduces TSNAs by screening strains with weak nitrate degradation ability and strong nitrite degradation ability.

[0004] The present invention fully exploits the microbial resources in cigars, and reduces the main harmful substance in tobacco, TSNAs, through the research of functional strains derived from tobacco, thereby improving the safety of cigar smoking. In recent years, the tobacco industry has used microorganisms to reduce nitrite, a precursor of TSNAs, in sun-cured and flue-cured tobacco, but the research on cigar TSNA reduction technology is almost blank. The cigar fermentation process is a process with high participation of microorganisms. The microbial strains used by previous researchers to reduce TSNAs in the drying and aging stages of cigarette raw materials may not be suitable for the high-temperature fermentation process of cigars. Summary of the invention

[0005] To solve the above problems, the object of the present invention is to provide a thermostable Bacillus Velez subtilis strain, which can efficiently degrade nitrite and tobacco-specific nitrosamines.

[0006] The second object of the present invention is to provide the use of the Bacillus Velez in degrading nitrite and tobacco-specific nitrosamines.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A Bacillus velezensis strain, which is Bacillus velezensis DX32 and has a deposit number of CCTCC NO: M 2022908.

[0009] The invention separates and screens a thermostable Bacillus velezensis DX-32 from imported finished cigars, which can efficiently degrade nitrite and tobacco-specific nitrosamines.

[0010] Application of Bacillus velezensis in the degradation of nitrite and tobacco-specific nitrosamines in tobacco.

[0011] The Bacillus Velez is screened and isolated from the surface of cigar leaves. It is easier to grow on cigar leaves than general exogenous bacteria. It can efficiently degrade nitrite, the precursor of tobacco-specific nitrosamines, and can also efficiently degrade tobacco-specific nitrosamines. It is of great significance in improving tobacco safety and protecting the health of tobacco consumers.

[0012] Preferably, the tobacco is cigar tobacco.

[0013] Preferably, the tobacco-specific nitrosamines are NNN, NNK, NAB, and NAT. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The degradation ability of Bacillus velezensis DX-32 and the control bacteria WD-04 on NNN and NNK in Example 2 of the present invention is compared;

[0015] Figure 2 is a colony morphology diagram of Bacillus velezensis DX-32 on LB medium in Example 3 of the present invention;

[0016] Figure 3 is a 100× microscopic morphology picture of Bacillus velezensis DX-32 in Example 3 of the present invention;

[0017] Figure 4 is the phylogenetic tree constructed based on the 16SrDNA gene sequence of Bacillus velezensis DX-32 in Example 3 of the present invention;

[0018] Figure 5 is a comparison chart of the ability of Bacillus velezensis DX-32 to degrade NNN in different concentrations of NNN culture medium in Example 4 of the present invention;

[0019] Figure 6 This is the degradation ability of Bacillus velezensis DX-32 on TSNAs during the stacking fermentation of cigar tobacco leaves in Example 5 of the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0021] Some of the biological materials, experimental reagents, experimental equipment, etc. involved in the following embodiments and test examples are briefly introduced as follows:

[0022] Culture medium:

[0023] Nitrite enrichment medium: NaNO2 0.5 g / L, MgSO4·7H2O 0.2 g / L, K2HPO4 0.5 g / L and KNaC4H4O6·4H2O 20.0 g / L; pH 7.2, sterilization at 121℃ for 30 min.

[0024] Nitrite separation medium: Add 15.0 g / L agar powder to the enrichment medium.

[0025] Nitrate culture medium: NaNO3 2.5g / L, MgSO4·7H2O 0.2g / L, K2HPO4 0.5g / L and KNaC4H4O6·4H2O20.0g / L; pH 7.2~7.4, sterilization at 121℃ for 30min.

[0026] Nitrate separation medium: Add 15.0 g / L agar to nitrate enrichment medium, pH 7.2-7.4, and sterilize at 121°C for 30 min.

[0027] NNN culture medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast powder, pH 7.2-7.4, sterilized at 121°C for 30 min. After the culture medium is sterilized, NNN is filtered and sterilized with a 0.22 μm filter membrane and then added at a content of 10 ppm.

[0028] NNK culture medium: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, pH 7.2-7.4, sterilized at 121°C for 30min. After the culture medium is sterilized, NNK is filtered and sterilized with a 0.22μm filter membrane and then added at a content of 10ppm.

[0029] NAT culture medium: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, pH 7.2-7.4, sterilization at 121℃ for 30min. After the culture medium is sterilized, NAT is filtered and sterilized with a 0.22μm filter membrane and then added at a content of 10ppm.

[0030] NAB medium: sodium chloride 10g / L, peptone 10g / L, yeast powder 5g / L, pH 7.2-7.4, sterilized at 121°C for 30min. After the medium is sterilized, NAB is filtered and sterilized with a 0.22μm filter membrane and then added at a content of 10ppm.

[0031] 1. A strain of Bacillus Velezii that efficiently degrades nitrite and TSNAs

[0032] A strain of Bacillus velezensis that efficiently degrades nitrite and TSNAs, the strain is Bacillus velezensis (Bacillus velezensis) DX-32, the preservation number is CCTCC NO: M 2022908; preservation date: June 16, 2022; preservation unit: China Center for Type Culture Collection, preservation address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0033] Example 1 Isolation and screening of strains capable of degrading nitrite and nitrate

[0034] This example separates and screens strains with strong nitrite degradation capabilities and weak nitrate degradation capabilities. The specific operations are as follows:

[0035] 1. Sample screening

[0036] According to the production of handmade cigar products and cigar tobacco leaves of major domestic and foreign brands and specifications, and considering the differences in the quality of different cigarettes and tobacco samples, different varieties and different years of tobacco leaves from different countries, representative handmade cigars and cigar tobacco leaf samples were selected and collected, and tobacco leaf samples to be fermented for the first time from representative domestic production areas were collected and refrigerated for standby use; at the same time, strains that can efficiently degrade nitrogen-containing compounds were selected from food source bacterial resources as the screening objects of excellent strains. The names, types and sources of the screened samples are shown in Table 1.

[0037] Table 1

[0038]

[0039]

[0040] 2. Screening of strains with strong ability to degrade nitrite

[0041] Take 10g of the above-mentioned cigar products and cigar tobacco leaves, add 100mL of sterile water for homogenization, and culture at 37℃, 180r / min for 3h. Take the supernatant and heat it in an 80℃ water bath for 15min to retain the heat-resistant strains. Take 1.5mL of the heated supernatant and add it to 50mL of liquid nitrite enrichment medium. Carry out targeted enrichment culture at 37℃, 180r / min for 2d. Take the supernatant in the enrichment culture and dilute it to 10 times. -3 , 10 -4 , 10 -5 Spread onto a nitrite separation medium plate with nitrite as the sole nitrogen source, culture at 37°C for 48 hours, isolate a single colony, select a single colony and streak it onto an LB plate, culture at 37°C for 24 hours, and store.

[0042] The isolated and preserved strains were selected and inoculated into liquid LB, cultured at 37°C, 180r / min for 12h as seed solution. The seed solution was inoculated into a liquid enrichment medium containing nitrite as the sole nitrogen source at a 3% (V / V) inoculation rate for 48h at 37°C, 180r / min, and CK was used as a control without inoculation. The bacterial solution was centrifuged at 10000r / min for 10min, and the supernatant was taken to detect the nitrite content in the supernatant.

[0043] 3. Determination of nitrite

[0044] Drawing of nitrite standard curve: After drying NaNO2 in an oven to constant weight, weigh 100 mg NaNO2, add water to make up to 100 mL, dilute 100 times to obtain a concentration of 10 μg / mL, then take 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL of sodium nitrite standard solution, transfer them to a 10 mL volumetric flask, make up to volume with distilled water, then take 2 mL of nitrite solution, add 4 mL of 1% sulfonamide, and then add 4 mL of 0.02% α-naphthylamine, react at 25 ° C for 30 min, and measure the absorbance at a wavelength of 540 nm on a UV-visible spectrophotometer with the blank (i.e., 0 mL of the standard solution) as the reference solution.

[0045] Determination of nitrite in the sample: Take 2 mL of the supernatant obtained by centrifugation after fermentation, add 4 mL of 1% sulfonamide, then add 4 mL of 0.02% α-naphthylamine, react at 25°C for 30 min, and detect the nitrite degradation rate of the screened strain at a wavelength of 540 nm on a UV-visible spectrophotometer. The nitrite degradation rate of the screened strain DX-32 is shown in Table 2. WD-04 in Table 2 is a Bacillus Velez strain screened from the same batch.

[0046] Table 2 Nitrite degradation rate of strain DX-32 screened

[0047] Strain number Nitrite degradation rate WD-04 39.62％ DX-32 98.26％

[0048] 4. Screening of strains with weak nitrate degradation ability

[0049] Since nitrate is reduced to nitrite, strains with strong nitrate-degrading ability have higher nitrate reductase activity in their bodies, which leads to its conversion into nitrite. Therefore, it is necessary to screen strains with weak nitrate-degrading ability among strains with strong nitrite-degrading ability.

[0050] Select the above-mentioned strains with strong nitrite degradation ability, streak inoculate on nitrate separation medium plates, culture at 37°C for 24 hours, and store. Pick the separated and stored strains and inoculate them in liquid LB, culture at 37°C, 180r / min, and culture for 12 hours as seed liquid. Inoculate the seed liquid into nitrate medium at an inoculum of 3% (V / V) for culture, 37°C, 180r / min, ferment and culture for 48 hours, CK is the control without inoculation. Centrifuge the bacterial solution at 10000r / min for 10 minutes, then take the supernatant and detect the nitrate content in the supernatant.

[0051] 5. Determination of nitrate

[0052] Drawing of nitrate standard curve: After drying NaNO3 in an oven to constant weight, weigh 100 mg NaNO3 and add water to make up to 100 mL. Take 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of sodium nitrate standard solution respectively, transfer to a 10 mL volumetric flask, and make up to volume with distilled water. Then take 0.1 mL of nitrate solution respectively, add 0.4 mL of 5% salicylic acid, let stand at room temperature for 20 min, then add 8% NaOH, transfer to a 10 mL volumetric flask, make up to volume with distilled water, let stand to room temperature, and measure the absorbance at a wavelength of 420 nm on a UV-visible spectrophotometer with blank (i.e., 0 mL of standard solution) as the reference solution.

[0053] Determination of nitrate in samples: Take 0.1 mL of the supernatant after fermentation centrifugation, add 0.4 mL of 5% salicylic acid, let stand at room temperature for 20 min, then add 8% NaOH, transfer to a 10 mL volumetric flask, make up to volume with distilled water, let stand to room temperature, detect its absorbance at a wavelength of 420 nm on a UV-visible spectrophotometer, and calculate the nitrate degradation rate, as shown in Table 3.

[0054] Table 3 Nitrate degradation rate of strain DX-32 screened

[0055] Strain number Nitrate degradation rate WD04 91.96％ DX-32 31.49％

[0056] According to the comprehensive analysis of the results of nitrate degradation rate and nitrite degradation rate, the DX-32 strain has a strong ability to degrade nitrite, but a weak ability to degrade nitrate.

[0057] Example 2 Screening and evaluation of strains capable of degrading tobacco-specific nitrosamines (TSNAs)

[0058] The strain DX-32 with strong nitrite degradation ability and weak nitrate degradation ability was streaked onto an LB plate and cultured at 37°C for 12 h. A single colony was picked and transferred to 5 mL of liquid LB medium and cultured at 37°C, 180 r / min, for 12 h as seed solution. The seed solution was inoculated at an inoculum amount of 3% (V / V) into 5 mL of NNN (NNN 10 ppm) medium, 5 mL of NNK (NNK10 ppm) medium, 5 mL of NAT (NAT 10 ppm), and 5 mL of NAB (NAB 10 ppm) medium, respectively, and cultured at 37°C, 180 r / min, for 72 h. CK was a control without inoculation of bacteria. The bacterial solution was centrifuged at 10000r / min for 5min, and the supernatant was taken. The contents of NNN, NNK, NAT and NAB were detected by HPLC-MS / MS. The degradation rates of NNN, NNK, NAT and NAB were calculated, and the degradation abilities of Bacillus Velezii DX-32 on various components of TSNAs were compared.

[0059] HPLC conditions: mobile phase A: aqueous solution, mobile phase B: methanol solution, flow rate 0.8 mL / min, column temperature 40°C, injection volume 10 μL, detection wavelength 235 nm, chromatographic column Poroshell EC-C18 (4.6*250 mm 4 um). Table 4 below shows the conditions for gradient elution.

[0060] Table 4 HPLC gradient elution conditions

[0061] Time (min) Flow rate (mL / min) A(％) B(％) 0.00 0.8 90 10 10.0 0.8 10 90 14.00 0.8 10 90 19.00 0.8 90 10 25.00 0.8 90 10

[0062] The contents of NNN, NNK, NAT and NAB in WD-04 (same strain) and DX-32 samples were calculated by HPLC, and the degradation rates of NNN, NNK, NAT and NAB were calculated. Figure 1 From the figure, it can be seen that the degradation rate of DX-32 strain for 10ppm NNN, the key component of TSNAs, is 21.07%; the degradation rate for 10ppm NNK, the key component of TSNAs, is 27.43%; the degradation rate for 10ppm NAT, the key component of TSNAs, is 35.69%; the degradation rate for 10ppm NNK, the key component of TSNAs, is 9.23%, which has a significant advantage over the control strain WD-04.

[0063] Example 3 Identification of Bacillus Velez DX-32

[0064] 1. Morphological identification

[0065] The DX-32 obtained from the above screening was inoculated onto LB solid culture medium plates and cultured at 37°C for 12 hours before observing the colony morphology. Figure 2 , Figure 3 As shown, the bacteria of Bacillus Velez DX-32 are straight rod-shaped. On LB solid medium, the colonies are round with uneven edges, and the surface of the bacterial lawn is dull, opaque, spreading, and milky white mucus.

[0066] 2. Molecular Biology Identification

[0067] The Velez Bacillus DX-32 strain was inoculated into LB medium, cultured at 180 rpm / min, 37°C, shaken for 12 hours, and the cells were collected by centrifugation. The 16S rDNA gene was amplified by PCR using the 16S rDNA universal primers forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR conditions were: 95°C, 5min; 95°C, 30s, 55°C, 30s, 72°C, 90s, 30 cycles; 95°C, 40s, 55°C, 45s, 72°C, 1.5min, 30 cycles; 72°C, 5min, 10°C, 5min. The PCR product was 1469bp in length, and its nucleotide sequence was shown in the sequence table SEQ ID NO: 1.

[0068] The 16S rDNA gene fragment of strain DX-32 was amplified and purified by PCR, and the DNA sequencing was completed by Beijing Qingke Biotechnology Co., Ltd. The measured sequence was analyzed for sequence similarity using the Blastn program of NCBI, and the 16S rDNA gene sequences of representative strains of similar species were selected from the Genbank database and the ribosome database. Phylogenetic analysis was performed using MEGA7.0 software, and a phylogenetic tree based on the 16S rDNA gene sequence was constructed using the neighbor joining method. The number of repeated samplings for testing the bootstrap support rate was 1000 times. The 16S rDNA gene sequence of the NNN efficient degradation bacteria DX-32 obtained by sequencing has 1469 bases. The results of homology analysis showed that the 16S rDNA gene sequence of strain DX-32 was highly similar to that of Bacillus velezensis, and a phylogenetic tree was constructed using MEGA7.0 software. The phylogenetic tree is shown in the figure. Figure 4As shown, it was shown that strain DX-32 belongs to Bacillus velezensis. Combining morphological characteristics and 16S rDNA gene sequence analysis, the strain was identified as Bacillus velezensis and named Bacillus velezensis DX-32. It was deposited in the China Center for Type Culture Collection (CCTCC) on June 16, 2022, with the deposit number CCTCC NO: 2022908.

[0069] 2. Application of Bacillus Velezii DX-32 in Degradation of Nitrite and TSNAs

[0070] Example 4 Degradation rate of NNN at different concentrations by Bacillus Velez DX-32

[0071] The screened Bacillus Velezii DX-32 was used to degrade NNN, the main carcinogen in TSNAs. The test results proved that DX-32 can effectively degrade NNN.

[0072] Take the Velez Bacillus DX-32 strain and streak it on the LB plate, culture it at 37℃ for 12h, pick a single colony and transfer it to 5mL liquid LB medium, culture it at 37℃, 180r / min, and culture it for 12h as seed liquid, inoculate the seed liquid into 5mL medium with different concentrations of NNN (NNN 100ppm, NNN 50ppm, NNN 10ppm) at an inoculum volume of 3% (V / V), and culture it at 37℃, 180r / min, and culture it for 36h. CK is the control without inoculation. The bacterial liquid was centrifuged at 10000r / min for 5min, and then the supernatant was taken to detect the degradation rate of NNN, and the degradation ability of Velez Bacillus DX-32 on different concentrations of NNN was compared. The results are as follows: Figure 5 As shown, compared with the control, the degradation rate of NNN, the key component of TSNAs, was the highest at 10 ppm, which was 29.56%.

[0073] Example 5: Analysis of the ability of Bacillus Velez DX-32 to degrade TSNAs in cigar tobacco leaves

[0074] The screened strain DX-32 with high efficiency in degrading nitrite and TSNAs was used to carry out laboratory simulation of cigar agricultural pile fermentation on CX-7, a representative cigar tobacco variety in Hubei Province.

[0075] Streak DX-32 onto an LB plate and culture at 37°C for 12 h. Pick a single colony and transfer it to 5 mL of liquid LB medium. Culture it at 37°C, 180 r / min, and transfer it to 100 mL of liquid LB medium as seed solution. Culture it at 37°C, 180 r / min, and culture it for 12 h. Spray the seed solution evenly on the leaves of cigar tobacco CX-7 at an inoculation rate of 50% (V / V). CK is a control without inoculation. Culture the leaves in a compacted pile at 37°C incubator for 96 h.

[0076] When fermentation reached 96h, cigar tobacco leaves were taken, dried at 100℃ for 3h, ground, passed through a 40-mesh sieve, and sampled. The contents of NNN, NNK, NAT, and NAB in fermented cigar tobacco leaves were detected according to YQ / T29-2013 "Determination of tobacco-specific N-nitrosamines in tobacco and tobacco products by high performance liquid chromatography-tandem mass spectrometry". The degradation rate of each component was calculated, and the degradation ability of DX-32 on TSNAs during the stacking fermentation of cigar tobacco leaves was analyzed. Figure 6 .

[0077] Through the detection and analysis of high performance liquid chromatography-tandem mass spectrometry, the degradation ability of cigar tobacco leaves treated with DX-32 strain (experimental group) and cigar tobacco leaves treated with equal amount of sterile water (control group) on various components of TSNAs during 96 hours of stacking fermentation is shown in Table 5.

[0078] Table 5 Analysis of degradation of TSNAs components of representative cigar tobacco leaves by DX-32

[0079] sample NNN NNK NAT NAB Total TSNAs Control group 2.79mg / kg 6.64mg / kg 5.24mg / kg 0.77mg / kg 15.44mg / kg Experimental Group 1.84mg / kg 3.65mg / kg 3.30mg / kg 0.56mg / kg 9.35mg / kg Degradation rate 34.25％ 45.01％ 36.93％ 27.94％ 39.47％

[0080] It can be seen from the above table that the degradation rates of NNN, NNK, NAT, NAB and total TSNAs by DX-32 during the stacking fermentation of cigar tobacco leaves were 34.25%, 45.01%, 36.93%, 27.94% and 39.47% respectively at 96h, which has good application value.

[0081] In summary, the Velez Bacillus DX-32 of the present invention can not only effectively reduce the content of nitrite, but also has a high degradation ability for each component of TSNAs, NNN, NNK, NAT and NAB, and also shows good degradation performance in the actual application of cigars. The bacterium has great application potential in reducing the harm of cigar tobacco leaves and their products, and can lay a foundation for the subsequent optimization of bacterial fermentation process and the improvement of cigar tobacco agricultural fermentation technology.

Claims

1. A strain of Bacillus velezine that degrades nitrite and tobacco-specific nitrosamines ( Bacillus velezensis ), characterized in that: The strain is Bacillus Velez Bacillus velezensis )DX-32, the accession number is CCTCCNO: M 2022908.

2. A Bacillus Velezii as claimed in claim 1 ( Bacillus velezensis ) in the degradation of nitrite and tobacco-specific nitrosamines in tobacco, characterized by: The tobacco-specific nitrosamines are N-nitrosonornicotine, 4-(N-nitrosomethyl nitrogen)-1-(3-pyridyl)-1-butanone, N-nitrosoanabasine and N-nitrosoanatabine.

3. The Bacillus Velezii according to claim 2 ( Bacillus velezensis ) in the degradation of nitrite and tobacco-specific nitrosamines in tobacco, characterized by: The tobacco is cigar tobacco leaves.

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