CN117187100A - Bacillus brevis Ni18 for efficiently degrading nicotine and tobacco specific nitrosamine and application thereof - Google Patents
Bacillus brevis Ni18 for efficiently degrading nicotine and tobacco specific nitrosamine and application thereof Download PDFInfo
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- 241000208125 Nicotiana Species 0.000 title claims abstract description 80
- 229960002715 nicotine Drugs 0.000 title claims abstract description 55
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 title claims abstract description 54
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 241000193764 Brevibacillus brevis Species 0.000 title claims abstract description 50
- 230000000593 degrading effect Effects 0.000 title claims abstract description 18
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- 150000004005 nitrosamines Chemical class 0.000 claims description 14
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a bacillus brevis Ni18 polluted by efficiently degrading nicotine and tobacco specific nitrosamine and application thereof, belonging to the field of microorganisms. The strain separated and screened from the surface of cigar tobacco leaves is identified as the bacillus brevis (Brevibacillus invocatus) to be polluted by morphology, molecular biology and the like, and is named as the bacillus brevis (Brevibacillus invocatus) Ni18 to be polluted, and the preservation number is CCTCC NO: m20221336. The strain can realize the efficient degradation of nicotine and specific nitrosamine in tobacco, has good stress resistance, is simple, convenient and easy to obtain, is convenient to use, has wide market application prospect, and provides a precious microbial resource for tobacco agriculture harm reduction.
Description
Technical Field
The invention relates to a bacillus brevis Ni18 polluted by efficiently degrading nicotine and tobacco specific nitrosamine and application thereof, belonging to the field of microorganisms.
Background
Nicotine, commonly known as Nicotine (Nicotine), is an alkaloid in common tobacco and also an important component of tobacco, and its effect is mainly due to the physiological strength, so-called stiffness, produced by it, which is proportional to the Nicotine content. The intake of nicotine brings excitation stimulus to the central nervous system of a person, and proper intake can lead the person to quickly refresh the spirit and weaken the fatigue and discomfort. However, excessive intake of nicotine may cause dizziness and vomiting in smokers, and may cause death in severe cases. Thus, excessive levels of nicotine in tobacco can be a serious hazard to smokers' health, and can reduce smoking-induced diseases by reducing the levels of nicotine.
Tobacco Specific Nitrosamines (TSNAs) are tobacco specific N-nitrosamines, a harmful substance present in tobacco, and are also one of the main harmful substances affecting human health. Of the 8 TSNAs that have been identified so far, 4 species of N-nitrosonornicotine (NNN), 4- (N-methyl-nitrosamine) -1- (3-pyridyl) -1-butanone (NNK), N-nitrosonew tobacco base (NAT) and N-Nitrosopseudoscouring (NAB) are higher in content. As early as 1962, the potential carcinogenesis with tobacco specific nitrosamines was first reported internationally, resulting in tumor formation in the lung, mouth, esophagus, stomach, pancreas, liver, etc. of humans. Therefore, the reduction of tobacco nitrosamines is important for improving the quality and safety of tobacco, and is a recent research hotspot.
Tobacco nicotine and nitrite are precursor substances for TSNAs formation, both synergistically affecting tobacco TSNAs content. Thus, reducing the levels of tobacco nicotine and nitrite helps reduce the production of TSNAs. Research shows that TSNAs is extremely low in fresh tobacco leaves harvested in maturity and is mainly generated and accumulated in the tobacco leaf modulating process. Although the former have explored methods for reducing TSNAs in tobacco leaves in many ways, many of them involve flue-cured tobacco, cigarettes, etc., including the use of equipment, tobacco conditioning, improved processes, etc., the overall effect is not quite obvious. Cigar tobacco leaves are subjected to special preparation and fermentation procedures, so that the cigar tobacco leaves have higher nicotine and TSNAs content than other tobacco types, and few methods for degrading cigar nitrosamine are reported.
Disclosure of Invention
In order to solve the problems, the first object of the invention is to provide a bacillus brevis (Brevibacillus invocatus) Ni18 pollution which can efficiently degrade nicotine and tobacco specific nitrosamines, wherein the bacillus brevis pollution is a strain which can efficiently degrade nicotine and tobacco specific nitrosamines, and has very important significance for improving tobacco quality and reducing harm of tobacco specific nitrosamines to human health and living environment.
A second object of the invention is to provide the use of Bacillus brevis (Brevibacillus invocatus) contaminated with tobacco specific nitrosamine degradation.
A third object of the present invention is to provide the use of Bacillus brevis (Brevibacillus invocatus) contaminated for the preparation of tobacco specific nitrosamine degradation products
The fourth object of the present invention is to provide a microbial preparation.
A fifth object of the present invention is to provide the use of a microbial preparation for degrading tobacco specific nitrosamines.
A sixth object of the invention is to provide the use of Bacillus brevis (Brevibacillus invocatus) for the degradation of nicotine.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the strain is a pollution bacillus brevis (Brevibacillus invocatus) Ni18 for efficiently degrading nicotine and tobacco specific nitrosamine, and is pollution bacillus brevis (Brevibacillus invocatus) Ni18, and is preserved in China Center for Type Culture Collection (CCTCC) NO: m20221336.
The invention separates and screens a strain of high-temperature-resistant pollution bacillus brevis (Brevibacillus invocatus) Ni18 from cigars, which can degrade nicotine and tobacco specific nitrosamines with high efficiency, and has great significance for improving tobacco quality and reducing harm of the tobacco specific nitrosamines to human health and living environment.
The application of the pollution Brevibacillus brevis (Brevibacillus invocatus) in the degradation of tobacco-specific nitrosamines.
The polluted bacillus brevis Ni18 is separated from the surface of cigar tobacco leaves, and can grow on the cigar tobacco leaves more easily than common exogenous bacteria; and the nicotine can be efficiently degraded by screening the nicotine from a culture medium with the nicotine as the only nitrogen source, and the method has important significance in improving the safety of tobacco and protecting the health of tobacco consumers.
Preferably, the tobacco is cigar tobacco.
Preferably, the tobacco specific nitrosamine is N-nitrosonornicotine, 4- (N-nitrosomethylnitrogen) -1- (3-pyridyl) -1-butanone, N-nitrosopseudoscouring, N-nitrosoneonicotinoid.
The application of the pollution Brevibacillus brevis (Brevibacillus invocatus) in preparing a tobacco-specific nitrosamine degradation product.
The polluted bacillus brevis Ni18 can efficiently degrade TSNAs, and can be used for preparing TSNAs degradation products for TSNAs degradation.
A microbial preparation comprising said Brevibacillus brevis (Brevibacillus invocatus) Ni18 contaminated.
The bacterial preparation can realize high-efficiency pollution-free degradation of nicotine and TSNAs, and is used for degrading nicotine in tobacco without damaging original good quality of tobacco, and increasing smoke of tobacco.
The application of the microbial preparation in degrading tobacco specific nitrosamines is provided.
The microbial preparation can be used for degrading TSNAs in tobacco, and provides a new direction for biodegradation of TSNAs in cigars.
The application of the pollution Brevibacillus brevis (Brevibacillus invocatus) in the aspect of nicotine degradation.
The polluted bacillus brevis (Brevibacillus invocatus) Ni18 is screened out by taking nicotine as a unique nitrogen source, so that the nicotine can be effectively degraded, and the degradation process is green and pollution-free.
Drawings
FIG. 1 is a comparison of the ability of Bacillus brevis (Brevibacillus invocatus) Ni18 contaminated in example 2 of the present invention to degrade NNN, NNK, NAB, NAT after fermentation;
FIG. 2 is a colony morphology of the contaminated Brevibacillus brevis (Brevibacillus invocatus) Ni18 on LB medium in example 3 of the present invention;
FIG. 3 is a photomicrograph (100X) of the Ni18 of Bacillus brevis (Brevibacillus invocatus) contaminated in example 3 of the present invention;
FIG. 4 is a mass spectrum fingerprint and matching bar chart of the invention in example 3 of the identification of Bacillus brevis (Brevibacillus invocatus) Ni18 using MALDI-TOF MS;
FIG. 5 is a phylogenetic tree of the Bacillus brevis (Brevibacillus invocatus) Ni18 contaminated with 16S rDNA gene sequence construction in example 3 of the present invention;
FIG. 6 shows the degradation rate of the TSNAs components of the cigar tobacco CX-81 by the Bacillus brevis (Brevibacillus invocatus) Ni18 contaminated in example 4 of the present invention;
FIG. 7 shows the degradation rate of the TSNAs components of the cigar tobacco CX-84 by the contaminated Bacillus brevis (Brevibacillus invocatus) Ni18 in example 4 of the present invention.
Detailed Description
The invention is further described in connection with the following detailed description, but the scope of the invention is not limited thereto; unless otherwise specified, all reagents, instruments, etc. used in the examples are commercially available.
The following examples and experimental examples are briefly described below for some of the biological materials, experimental reagents, experimental facilities, and the like:
culture medium:
nicotine enrichment medium: k (K) 2 HPO 4 3H 2 O 13.3g/L，KH 2 PO 4 4g/L，MgSO 4 7H 2 O0.2 g/L,0.5mL/L trace element solution. After sterilization of the medium, nicotine was added at a level of 1.5g/L after filtration sterilization with a 0.22 μm filter.
Trace element solution: caCl (CaCl) 2 ·2H 2 O 0.05g/L，CuCl 2 ·2H 2 O 0.05g/L，MnSO 4 ·H 2 O 0.008g/L，FeSO 4 ·7H 2 O 0.004g/L，ZnSO 4 ·7H 2 O 0.1g/L，Na 2 MoO 4 ·2H 2 O0.1 g/L was dissolved with a small amount of 0.1mol/L HCl.
Nicotine isolation medium: 15.0g/L agar powder is added into the enrichment medium.
LB medium: 10g/L of sodium chloride, 10g/L of peptone and 5g/L of yeast powder.
NNN medium: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder, pH 7.2-7.4, sterilizing for 30min at 121 ℃, sterilizing a culture medium, filtering and sterilizing NNN by using a 0.22 mu m filter membrane, and adding the NNN according to the final content of 10 mg/L.
NNK medium: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder, pH 7.2-7.4, sterilizing for 30min at 121 ℃, sterilizing a culture medium, filtering and sterilizing NNK by using a 0.22 mu m filter membrane, and adding the NNK according to the final content of 10 mg/L.
NAT medium: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder, pH 7.2-7.4, sterilizing for 30min at 121 ℃, sterilizing a culture medium, filtering and sterilizing by using a 0.22 mu m filter membrane, and adding the NAT according to the final content of 10 mg/L.
NAB medium: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder, pH 7.2-7.4, sterilizing for 30min at 121 ℃, sterilizing a culture medium, filtering and sterilizing NAB by a 0.22 mu m filter membrane, and adding the NAB according to the final content of 10 mg/L.
1. Brevibacillus brevis capable of efficiently degrading nicotine and TSNAs
The strain is a bacillus brevis (Brevibacillus invocatus) Ni18 strain for efficiently degrading nicotine and TSNAs, and the preservation number is CCTCC NO: m20221336; preservation date: 2022, 8, 26; preservation unit: china center for type culture collection, preservation address: eight branches of 299 Wuhan university in Wuhan district of Wuhan, hubei province.
EXAMPLE 1 isolation and screening of Nicotine-degrading Strain
The method for separating and screening the bacterial strain with strong nicotine degradation capability comprises the following specific operations:
1. sample screening
According to the production conditions of manual cigar products and cigar tobacco leaves of main brands and specifications at home and abroad, taking the differences of tobacco leaf sample quality, tobacco leaf sample quality and tobacco leaf sample quality of different varieties and tobacco leaf sample of different years into consideration, selecting and collecting representative manual cigar and cigar tobacco leaf samples, collecting and refrigerating tobacco leaf samples to be primarily fermented in representative domestic places for later use; the sample names, types and sources screened are shown in Table 1 below.
TABLE 1 Screen sample sources
2. Primary screen
Cutting cigar tobacco leaves of different varieties and regions at home and abroad, adding sterile water, oscillating at room temperature for extraction for 3h to obtain an extraction suspension, centrifuging, taking supernatant, heating in water bath at 60deg.C for 30min, and directionally retaining heat-resistant strain. Adding heated supernatant into enrichment culture medium with nicotine as sole carbon and nitrogen source, and enrichment culturing for 2d. And separating the strain which can grow on the nicotine separation medium by taking the centrifuged bacterial suspension and using a dilution plate method. And (3) streaking and purifying the screened strain by using an LB culture medium plate to obtain a single colony with vigorous growth.
3. Double screen
The separated and preserved strain is selected and inoculated into liquid LB, and is cultured for 12 hours at 37 ℃ to be used as seed liquid. The seed solution was inoculated into a nicotine fermentation medium having an initial concentration of 1.5g/L at an inoculum size of 3% (V/V), and incubated at 37℃for 24 hours, as a control without inoculating bacteria. And centrifuging the bacterial liquid to obtain supernatant, and detecting the nicotine content in the supernatant by using an ultraviolet spectrophotometry at 72h, wherein the principle is that potassium permanganate and nicotine react in NaOH solution to form a water-soluble green product, and the green product has a maximum absorption value at 610 nm. The test is completed and the nicotine degradation rate is calculated:
nicotine degradation rate = (nicotine content in stock culture-nicotine content in fermentation broth)/nicotine content in stock culture × 100%
The strain which can utilize the growth of nicotine and is obtained by primary screening is fermented, and further screening is carried out to obtain a strain Ni18 which can efficiently degrade nicotine, and the nicotine degradation rate of the strain which can efficiently degrade nicotine is obtained by secondary screening is shown in Table 2. M8 is the strain screened in the same batch.
TABLE 2 degradation Rate of highly efficient degradation of Nicotine Strain
| Strain numbering | Nicotine degradation rate |
| M8 | 22.47％ |
| Ni18 | 53.76％ |
EXAMPLE 2 screening and evaluation of tobacco-specific nitrosamine (TSNAs) degrading Strain
Inoculating the strain Ni18 with strong nicotine degrading capability to LB culture medium, culturing at 37 ℃ for 12h to obtain fermentation seeds, inoculating the fermentation seeds to liquid LB culture medium, culturing at 37 ℃ for 12h to obtain seed liquid, and inoculating the seed liquid to 5mL NNN culture medium (NNN 10 mg/L), 5mL NNK culture medium (NNK 10 mg/L), 5mL NAT culture medium (NAT 10 mg/L) and 5mL NAB culture medium (NAB 10 mg/L) respectively at an inoculum size of 3% (V/V) to perform culture, wherein the culture is carried out at 37 ℃ for 72h, and the culture is used as a control without inoculating bacteria. And centrifuging the fermented bacterial liquid to obtain supernatant, testing the content of NNN, NNK, NAT, NAB, calculating the degradation rate of NNN, NNK, NAT, NAB, and comparing the degradation capacity of the polluted Brevibacillus brevis Ni18 on each component of TSNAs.
The detection method of TSNAs comprises the following steps: HPLC conditions: mobile phase a: aqueous solution, mobile phase B: methanol solution with flow rate of 0.8mL/min, column temperature of 40deg.C, sample injection amount of 10 μl, detection wavelength of 235nm, and chromatographic column Porosill EC-C18 (4.6X250 mm4 um). Table 3 below shows the conditions for gradient elution.
TABLE 3HPLC gradient elution conditions
| 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 |
The degradation rate of NNN, NNK, NAT, NAB was calculated by HPLC analysis and the NNN, NNK, NAT, NAB content of the control sample and post-fermentation sample was calculated as shown in FIG. 1. From the graph, the degradation rate of 10mg/L NNN of the key component of TSNAs by Ni18 bacteria is 28.92%; the degradation rate of NNK of 10mg/L of key component of TSNAs is 37.21%; the degradation rate of NAT of 10mg/L for key components of TSNAs is 44.42%; the degradation rate of NNK of 10mg/L of key component of TSNAs is 25.97%, and compared with control strain, the degradation rate of NNK has obvious advantages.
EXAMPLE 3 identification of Brevibacillus contamination Ni18
1. Morphological identification
Ni18 obtained by the screening is inoculated on an LB solid medium plate, and colony morphology is observed after culture. As shown in FIG. 2 and FIG. 3, the contaminated Bacillus brevis Ni18 cells were in the form of short rods, and the colonies on the medium were round, pale yellow, smooth at the edges, moist and sticky.
2. MALDI-TOF MS identification
A single colony of the polluted Brevibacillus Ni18 is prepared, a strain suspension is obtained by using an extraction method, 1 mu L of sample is sucked to a target plate point, 1 mu L of matrix solution is dripped after the sample is dried, and a full-automatic microorganism mass spectrum detection system (Autof ms2000, anji Biotechnology Co., ltd.) is used for strain identification. Ni18 is identified as the bacillus brevis pollution by Autof ms2000, and a mass spectrum fingerprint and a matching bar chart are shown in figure 4.
3. Molecular biological identification
Inoculating the strain of the bacillus brevis Ni18 to a liquid LB culture medium for shake culture for 12 hours, centrifugally collecting thalli, and carrying out PCR amplification on a 16S rDNA gene by using a forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and a reverse primer 1492R (5'-GGTTACCTTGTTACGACTT-3') of the 16S rDNA universal primer, wherein the PCR conditions are as follows: 95 ℃ for 5min; circulation is carried out at 95 ℃,30s,55 ℃,30s,72 ℃,90s and 30; circulation at 95 ℃,40s,55 ℃,45s,72 ℃,1.5min, 30; 72 ℃,5min,10 ℃ and 5min. The length of the PCR product is 1393bp, and the nucleotide sequence table is shown as SEQ ID NO: 1.
The 16S rDNA gene fragment of the strain Ni18 is amplified by PCR, purified and recovered, and DNA sequencing work is completed by Beijing qing biological science and technology Co. The sequence similarity analysis was performed on the measured sequence by using the Blastn program of NCBI, the 16S rDNA gene sequence of a representative strain of similar species was selected from the Genbank database and the ribosome database, the phylogenetic analysis was performed by MEGA 7.0 software, and a phylogenetic tree based on the 16S rDNA gene sequence was constructed by the adjacency method (Neighbor training) for checking the Bootstrap support rate (Bootstrap) at 1000 times. The 16S rDNA gene sequence of the Ni18 obtained by sequencing has 1393 bases, and the homology analysis result shows that the 16S rDNA gene sequence of the strain Ni18 is highly similar to that of the bacillus brevis (Brevibacillus invocatus), and a phylogenetic tree is constructed by MEGA 7.0 software, and the phylogenetic tree is shown as figure 5, so that the strain Ni18 belongs to the bacillus brevis. Combining morphological characteristics and 16S rDNA gene sequence analysis, the strain is identified as the Bacillus brevis (Brevibacillus invocatus) Ni18, named as Bacillus brevis Ni18, and is preserved in China Center for Type Culture Collection (CCTCC) at the month of 2022 and the day of 8, with the preservation number of CCTCC NO: m20221336.
2. Application of pollution Brevibacillus Ni18 in degrading cigar tobacco TSNAs
EXAMPLE 4 Bacillus brevis contaminated Ni18 analysis of TSNAs degrading ability in cigar tobacco
Inoculating the polluted bacillus brevis Ni18 to an LB plate for activation culture for 12 hours, inoculating the activated polluted bacillus brevis Ni18 to a liquid LB culture medium, and fermenting for 12 hours in a shaking table at 37 ℃ and 180rpm to prepare a fermentation broth. The fermentation broth was centrifuged at 12000rpm for 5min, and the supernatant was removed to collect the cells. Resuspension the collected cells, measuring the cell concentration with a spectrophotometer, and diluting the cell concentration to 10 8 cfu/mL is the fermentation inoculant for polluting the Ni18 of the Brevibacillus. A certain amount of two cigar leaves (CX-81 and CX-84) with high TSNAs content are weighed, cut into triangular bottles by using sterile scissors to be used as a cigar leaf fermentation culture medium, so as to keep the total water content of the cigar leaves to be 60 percent, a certain amount of microbial inoculum is applied to the cigar leaves, the cigar leaves are uniformly sprayed on the tobacco leaves, and the cigar leaves are fermented for 5 days at 37 ℃ and RH75 percent.
After fermentation, taking cigar leaves, drying at 60 ℃ for 3 hours, grinding, sieving with a 60-mesh sieve, sampling, detecting NNN, NNK, NAT, NAB content in the cigar leaves after fermentation by YQ/T29-2013 'high performance liquid chromatography-tandem mass spectrometry combination method for measuring N-nitrosamine specific to tobacco and tobacco products', calculating degradation rate of each component, and analyzing degradation capability of polluted Bacillus brevis Ni18 on TSNAs of different types of cigar leaves with high TSNAs content.
The degradation capability of the polluted bacillus brevis Ni18 on each component of TSNAs in cigar tobacco leaves of different types is shown in the following table 4 through detection analysis of a high performance liquid chromatography-tandem mass spectrometry.
TABLE 4 Effect of Ni18 on different cigar tobacco types
Note that: CX-81 is Chuxue No. 81 cigar variety; CX-84 is Chuxue No. 84 cigar variety. CK represents the control group.
As shown in the table, ni18 has a strong degradation effect on TSNAs in cigar tobacco leaves with different types and high TSNAs content, wherein the degradation rate of NNK in CX-81 reaches 53%, the degradation rates of NNN and total TSNAs in CX-84 reach 53% and 46%, the degradation efficiency is shown in figures 6 and 7, and the application value is good.
In conclusion, the strain Ni18 provided by the invention can not only efficiently reduce the content of nicotine, but also has higher degradation capability on each component NNN, NNK, NAT, NAB of TSNAs, and also has better degradation capability in practical application of cigars. The strain has great application potential in reducing harm of cigar tobacco leaves and products thereof, and can lay a foundation for optimizing the subsequent microbial inoculum fermentation process and improving cigar fermentation.
The foregoing embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention, and other variations or modifications in light of the above teachings and concepts will be apparent to those skilled in the art and are not intended to be exhaustive or all embodiments of the invention. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (8)
1. A bacillus brevis (Brevibacillus invocatus) Ni18 polluted by efficiently degrading nicotine and tobacco-specific nitrosamine is characterized in that: the strain is pollution short bacillus (Brevibacillus invocatus) Ni18, and is preserved in China Center for Type Culture Collection (CCTCC) NO: m20221336.
2. Use of a contaminated bacillus brevis (Brevibacillus invocatus) as claimed in claim 1 for tobacco specific nitrosamine degradation.
3. Use of a contaminated bacillus brevis (Brevibacillus invocatus) according to claim 2 for the degradation of tobacco specific nitrosamines, characterized in that: the tobacco is cigar tobacco.
4. Use of a contaminated bacillus brevis (Brevibacillus invocatus) according to claim 2 for the degradation of tobacco specific nitrosamines, characterized in that: the tobacco specific nitrosamine is N-nitrosonornicotine, 4- (N-nitrosomethyl nitrogen) -1- (3-pyridyl) -1-butanone, N-nitroso pseudoscouring and N-nitroso neonicotinoid.
5. Use of a contaminated bacillus brevis (Brevibacillus invocatus) according to claim 1 for the preparation of a tobacco specific nitrosamine degradation product.
6. A microbial preparation, characterized in that: comprising the contaminated Brevibacillus brevis (Brevibacillus invocatus) Ni18 of claim 1.
7. Use of the microbial preparation of claim 6 for degrading tobacco specific nitrosamines.
8. Use of a contaminated bacillus brevis (Brevibacillus invocatus) according to claim 1 for nicotine degradation.
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