Bacillus flexus ni3, application and microbial bacterial preparation

Abstract

The present application relates to a kind of Bacillus flexus Ni3 and application and microbial bacteria preparation, belong to the field of microorganism.The present application is with nicotine as only carbon source and energy source to carry out preliminary screening and rescreening, then using TSNAs carries out degradation effect evaluation, separates a strain that can efficiently degrade nicotine and TSNAs Ni3, is identified as Bacillus flexus (Bacillus flexus) by morphology, MALDI-TOF MS and molecular biology identification.Bacillus flexus (Bacillus flexus) Ni3 has been preserved in China typical culture collection center, and the preservation number is CCTCC NO: M 20221334.Bacillus flexus (Bacillus flexus) Ni3 can efficiently degrade nicotine and TSNAs, has important significance in improving tobacco safety, protecting tobacco consumer health.

Description

Technical Field

[0001] This invention relates to a Bacillus curvatureis Ni3, its applications, and microbial preparations, belonging to the field of microbiology. Background Technology

[0002] Tobacco-specific nitrosamines (TSNAs) are N-nitroso compounds unique to tobacco. At the molecular level, they are complexes formed by the reaction of tobacco alkaloids and nitrosyl groups. They are present in both tobacco leaves and tobacco smoke, with eight types currently identified. The four most prevalent are N-nitrosonornicotinic acid (NNN), 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosopseudoesequine (NAB), and N-nitrosoneonicotinic acid (NAT). Studies have shown that NNN and NNK possess certain carcinogenic and mutagenic properties. NNK has also been identified as one of the most important indicators of harmful components affecting the toxicity of cigarette smoke. Therefore, reducing the content of TSNAs in tobacco and tobacco products is crucial to minimizing their harmful effects.

[0003] Tobacco leaves contain abundant amino compounds, including amino acids, proteins, and alkaloids. Nicotine is the most important alkaloid in tobacco, accounting for over 94% of the total alkaloid content. Other important but less abundant alkaloids include nornicotinic acid, neonicotinoids, and pseudoequisetine. It is generally believed that NNK originates from nicotine, NNN from nicotine and nornicotinic acid, NAT from neonicotinoids, and NAB from pseudoequisetine. Therefore, reducing the nicotine content in tobacco and tobacco products can help reduce TSNAs.

[0004] Among various tobacco products, cigars have the highest total TSNA content. The TSNA content in cigars is 1-2 orders of magnitude higher than that in blended and flue-cured cigarettes (Shi Hongzhi et al., 2002; Shi Hongzhi et al., 2012; Leng Hongqiong et al., 2011), with cigars sometimes exceeding 50 g / g (Hilldrup et al., 2019). Current research indicates that TSNAs are formed primarily through two pathways: direct nitrosation of alkaloids in tobacco and reaction between nitrites or nitrogen oxides and alkaloids. Nitrite is derived from the reduction of nitrates in tobacco leaves by microorganisms, thus it is generally believed that microorganisms play a crucial regulatory role in TSNA formation in tobacco leaves. In recent years, the tobacco industry has conducted some exploratory research and applications using microorganisms to reduce TSNA precursors in sun-cured and flue-cured tobacco, but research on TSNA reduction technology in cigars is almost nonexistent. Domestically produced cigars all employ a high-temperature, high-humidity fermentation process to ferment cigar tobacco leaves in bulk, which provides the possibility for the application of microbial inoculants. However, the unique bulk fermentation process of cigars involves a dramatic increase in temperature. Currently reported research suggests that strains that reduce TSNAs, based on applications in tobacco leaf preparation and cigarette processing, may not be suitable. Therefore, it is urgent to screen strains with strong high-temperature tolerance during cigar fermentation to maximize their effectiveness. Summary of the Invention

[0005] To address the aforementioned problems, the first objective of this invention is to provide a Bacillus curvaturelis (Bacillus) Bacillus bent Ni3, the strain being Bacillus curvatureis ( Bacillus flexus Ni3 can efficiently degrade nicotine and TSNAs in tobacco.

[0006] A second object of the present invention is to provide the aforementioned Bacillus curvatureus ( Bacillus flexus Applications in the degradation of tobacco-specific nitrosamines.

[0007] A third objective of this invention is to provide a microbial preparation.

[0008] A fourth objective of this invention is to provide the application of microbial preparations in the degradation of tobacco-specific nitrosamines in tobacco.

[0009] The fifth object of this invention is to provide Bacillus curvaturei ( Bacillus flexus Applications of nicotine degradation.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A type of Bacillus curvature ( Bacillus flexusNi3, the strain being Bacillus curvatureis ( Bacillus bent Ni3, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20221334.

[0011] Bacillus curvature can secrete various enzymes, such as pectinase and protease, making it a preferred microbial agent for improving the microbial environment. This invention isolates and screens a heat-resistant strain of Bacillus curvature from cigar smoke. Bacillus flexus Ni3 can efficiently degrade both nicotine and tobacco-specific nitrosamines, providing a new microbial resource for the degradation of nicotine and TSNAs.

[0012] The above-mentioned Bacillus curvatureis ( Bacillus flexus Applications in the degradation of tobacco-specific nitrosamines.

[0013] This Bacillus curvatureis, isolated from the surface of cigar tobacco leaves, grows more easily on cigar tobacco leaves than common exogenous bacteria. It can efficiently degrade nicotine, a precursor to tobacco-specific nitrosamines, and is of great significance in improving tobacco safety and protecting the health of tobacco consumers.

[0014] Preferably, the tobacco is cigar tobacco.

[0015] Preferably, the tobacco-specific nitrosamine is N-nitrosodemethylnicotinic acid, 4-(N-nitrosomethylnitrogen)-1-(3-pyridyl)-1-butanone, N-nitrosopseudoestiline, or N-nitrosoneonicotinic acid.

[0016] A microbial preparation comprising the aforementioned Bacillus curvatureis ( Bacillus flexus )Ni3.

[0017] This microbial preparation can achieve efficient and pollution-free degradation of TSNAs in tobacco.

[0018] Application of microbial inoculants in the degradation of tobacco-specific nitrosamines in tobacco.

[0019] Microbial preparations can efficiently and pollution-free degrade TSNAs in tobacco, which is of great significance for improving tobacco quality, reducing tobacco-specific nitrosamines, and maintaining human health.

[0020] The above-mentioned Bacillus curvatureis ( Bacillus flexus Applications of nicotine degradation.

[0021] Bacillus curvatureis Ni3 was screened and isolated using nicotine as the sole carbon source. It can effectively decompose nicotine and is of great significance in the degradation of nicotine in tobacco and tobacco waste. Attached Figure Description

[0022] Figure 1 It is the Bacillus curvatureis (Curvularia) in Example 1 of this invention. Bacillus flexus Comparison of the degradation capabilities of Ni3 after fermentation of NNN, NNK, NAB, and NAT; Figure 2 It is the Bacillus curvatureis (Curvularia) in Example 1 of this invention. Bacillus flexus Colony morphology of Ni3 on LB medium; Figure 3 It is the Bacillus curvatureis (Curvularia) in Example 1 of this invention. Bacillus flexus Ni3 microstructure (100×); Figure 4 It is the Bacillus curvatureis (Curvularia) in Example 1 of this invention. Bacillus flexus Mass spectrometry fingerprint and matching bar graph of Ni3 identified by MALDI-TOF MS; Figure 5 It is the Bacillus curvatureis (Curvularia) in Example 1 of this invention. Bacillus flexus A phylogenetic tree of Ni3 based on the 16S rDNA gene sequence; Figure 6 It is the Bacillus curvatureis (Curvularia) in Example 2 of this invention. Bacillus flexus The degradation rate of Ni3 on various components of TSNAs in CX-81 cigar tobacco leaves; Figure 7 It is the Bacillus curvatureis (Curvularia) in Example 2 of this invention. Bacillus flexus The degradation rate of Ni3 on the TSNAs components of CX-84 cigar tobacco leaves. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; unless otherwise specified, all reagents, instruments and other items used in the embodiments are commercially available products.

[0024] The following is a brief introduction to some of the biological materials, experimental reagents, and experimental equipment involved in the following examples and experimental cases: Culture medium: Nicotine enrichment medium: K₂HPO₄·3H₂O 13.3 g / L, KH₂PO₄ 4 g / L, MgSO₄·7H₂O 0.2 g / L, 0.5 mL / L trace element solution. After sterilization of the medium, nicotine was added at a concentration of 1.5 g / L after being sterilized by filtration through a 0.22 μm filter membrane.

[0025] Trace element solution: CaCl2·2H2O 0.05g / L, CuCl2·2H2O 0.05g / L, MnSO4·H2O 0.008g / L, FeSO4·7H2O 0.004g / L, ZnSO4·7H2O 0.1g / L, Na2MoO4·2H2O 0.1g / L, dissolved in a small amount of 0.1mol / L HCl.

[0026] Nicotine isolation medium: 15.0 g / L agar powder was added to the enrichment medium.

[0027] LB medium: sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L.

[0028] NNN medium: sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L, pH 7.2~7.4, sterilized at 121 ℃ for 30 min. After sterilization, NNN is filtered through a 0.22 μm filter membrane for sterilization and then added at a final concentration of 10 mg / L.

[0029] NNK medium: sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L, pH 7.2~7.4, sterilized at 121 ℃ for 30 min. After sterilization, NNK is filtered through a 0.22 μm filter membrane for sterilization and then added at a final concentration of 10 mg / L.

[0030] NAT medium: sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L, pH 7.2~7.4, sterilized at 121 ℃ for 30 min. After sterilization, NAT is filtered through a 0.22 μm filter membrane for sterilization and then added at a final concentration of 10 mg / L.

[0031] NAB medium: sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L, pH 7.2~7.4, sterilized at 121 ℃ for 30 min. After sterilization, NAB is filtered through a 0.22 μm filter membrane for sterilization and then added at a final concentration of 10 mg / L.

[0032] Example 1: A type of Bacillus curvatureus ( Bacillus flexus ) This embodiment of Bacillus curvatureis ( Bacillus flexus The bacteria were isolated from the surface of cigars and screened first and second times using nicotine as the sole carbon and energy source. The degradation effect was then evaluated using TSNAs. A bacterium, Ni3, capable of efficiently degrading nicotine and TSNAs, was isolated and identified as *Bacillus curvatureis* by morphological, MALDI-TOF MS, and molecular biological methods. Bacillus flexus The Bacillus curvatureus ( Bacillus flexusNi3, accession number CCTCC NO: M20221334; accession date: August 26, 2022; depositary institution: China Center for Type Culture Collection, address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province. The specific procedures for isolation, screening, evaluation, and identification are as follows: 1. Isolation and screening of nicotine-degrading strains 1.1 Sample Screening Based on the production status of handmade cigars and cigar tobacco leaves of major domestic and international brands and specifications, and considering the differences in the quality of different cigar and tobacco leaf samples from different countries, as well as the differences in the varieties and years of tobacco leaves, representative handmade cigar and cigar tobacco leaf samples were selected and collected. Samples of tobacco leaves awaiting primary fermentation from representative domestic production areas were also collected and refrigerated for later use. The names, types and sources of the screened samples are shown in Table 1 below.

[0033] Table 1 Sources of bacterial screening samples 1.2 Initial screening Cigar tobacco leaves of different varieties and regions from both domestic and international sources were chopped and added to sterile water. The mixture was extracted by shaking at room temperature for 3 hours to obtain an extract suspension. The supernatant was collected by centrifugation and heated in a 60°C water bath for 30 minutes to selectively preserve heat-resistant strains. The supernatant was then added to an enrichment medium with nicotine as the sole carbon and nitrogen source and cultured for 2 days. The centrifuged bacterial suspension was then used to isolate strains capable of growing on nicotine isolation medium using a dilution plate method. The selected strains were purified by streaking on LB agar plates to obtain vigorous single colonies.

[0034] 1.3 Secondary screening The isolated and preserved bacterial strain was inoculated into liquid LB and cultured at 37°C for 12 h to prepare the seed culture. The seed culture was then inoculated at a rate of 3% (V / V) into nicotine fermentation medium with an initial concentration of 1.5 g / L and cultured at 37°C for 24 h. An uninoculated culture served as a control. The supernatant was collected by centrifugation, and the nicotine content in the supernatant was determined using UV spectrophotometry at 72 h. The principle is that potassium permanganate reacts with nicotine in NaOH solution to form a water-soluble green product, which has a maximum absorbance at 610 nm. The nicotine degradation rate was calculated after the test. Nicotine degradation rate = (Nicotine content in original culture medium - Nicotine content in fermentation broth) / Nicotine content in original culture medium × 100% The strains that could utilize nicotine for growth obtained from the initial screening were fermented, and a further screening yielded a strain Ni3 that could efficiently degrade nicotine. The nicotine degradation rate of the strains obtained from the second screening is shown in Table 2. X5 is a strain screened from the same batch.

[0035] Table 2 Degradation rate of highly efficient nicotine-degrading strains 2. Evaluation of strains that degrade tobacco-specific nitrosamines (TSNAs) The strain Ni3, known for its strong nicotine-degrading ability, was inoculated onto LB medium and cultured at 37°C for 12 hours to obtain fermentation seed. This seed was then inoculated into liquid LB medium and cultured at 37°C for 12 hours to obtain seed culture. The seed culture was then inoculated at 3% (v / v) into 5 mL of NNN medium (NNN 10 mg / L), 5 mL of NNK medium (NNK 10 mg / L), 5 mL of NAT medium (NAT 10 mg / L), and 5 mL of NAB medium (NAB 10 mg / L) for 72 hours at 37°C. An uninoculated control was used. After fermentation, the bacterial culture was centrifuged, and the supernatant was collected. The contents of NNN, NNK, NAT, and NAB were tested, and the degradation rates of NNN, NNK, NAT, and NAB were calculated. The degradation abilities of *Bacillus curvatureis* Ni3 on various components of TSNAs were compared.

[0036] TSNAs detection method: HPLC conditions: mobile phase A: aqueous solution, mobile phase B: methanol solution, flow rate 0.8 mL / min, column temperature 40℃, injection volume 10 μL, detection wavelength 235 nm, chromatographic column Poroshell EC-C18 (4.6*250mm4um). Table 3 below shows the gradient elution conditions.

[0037] Table 3 HPLC gradient elution conditions HPLC analysis was used to determine the contents of NNN, NNK, NAT, and NAB in the control sample and the fermented sample, and the degradation rates of NNN, NNK, NAT, and NAB were calculated. Figure 1 The figure shows that strain Ni3 exhibited a 23.31% degradation rate of NNN (10 mg / L), a key component of TSNAs; a 64.10% degradation rate of NNK (10 mg / L), a 26.42% degradation rate of NAT (10 mg / L), a 27.42% degradation rate of NNK (10 mg / L), a key component of TSNAs. Compared with the control strain, it showed a significant advantage.

[0038] 3. Identification of Bacillus curvatureni Ni3 3.1 Morphological identification The Ni3 cells obtained from the above screening were inoculated onto LB solid medium plates, and colony morphology was observed after incubation. Results are as follows: Figure 2 , Figure 3As shown, Bacillus curvatureis Ni3 cells are rod-shaped, and the colonies on the culture medium are irregular in shape, pale yellow, and have transparent serrated edges.

[0039] 3.2 MALDI-TOF MS Identification Single colonies of *Bacillus curvatureis* Ni3 were prepared. A suspension of the strain was obtained using the extraction method. 1 μL of sample was pipetted onto the target plate, dried, and then 1 μL of matrix solution was added. The strain was identified using an automated microbial mass spectrometry system (Autof ms2000, Antu Biotechnology Co., Ltd.). Ni3 was identified as *Bacillus curvatureis* by Autof ms2000. The mass spectrometric fingerprint and matching bar graph are shown below. Figure 4 As shown.

[0040] 3.3 Molecular biological identification Bacillus curvaturenii strain Ni3 was inoculated into liquid LB medium and cultured with shaking for 12 hours. The cells were collected by centrifugation. PCR amplification of the 16S rDNA gene was performed using universal 16S rDNA primers: forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR conditions were: 95℃, 5 min; 95℃, 30 s, 55℃, 30 s, 72℃, 90 s, 30 cycles; 95℃, 40 s, 55℃, 45 s, 72℃, 1.5 min, 30 cycles; 72℃, 5 min, 10℃, 5 min. The PCR product was 1458 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 1.

[0041] The 16S rDNA gene fragment of strain Ni3 was amplified by PCR, purified, and recovered. DNA sequencing was performed by Beijing Qingke Biotechnology Co., Ltd. The obtained sequences were analyzed for sequence similarity using NCBI's Blasten program. Representative 16S rDNA gene sequences of closely related species were selected from Genbank and ribosome databases. Phylogenetic analysis was performed using MEGA 7.0 software. A phylogenetic tree based on the 16S rDNA gene sequence was constructed using the neighbor joining method. The number of replicates for bootstrap testing was 1000. The sequenced 16S rDNA gene sequence of Ni3 contained 1458 bases. Homology analysis showed that the 16S rDNA gene sequence of strain Ni3 was similar to that of *Bacillus curvatureis* (…). Bacillus flexus They are highly similar, and a phylogenetic tree was constructed using MEGA 7.0 software. The phylogenetic tree is as follows: Figure 5As shown, strain Ni3 belongs to Bacillus curvatureis. Based on morphological characteristics and 16S rDNA gene sequence analysis, this strain was identified as Bacillus curvatureis (…). Bacillus flexus The strain, named Bacillus curvatureis Ni3, was deposited at the China Center for Type Culture Collection (CCTCC) on August 26, 2022, with accession number CCTCC NO: M 20221334.

[0042] Example 2: Application of Bacillus curvatureis Ni3 in the degradation of tobacco-specific nitrosamines This embodiment describes the application of *Bacillus curvatureis* Ni3 in the degradation of nicotine and tobacco-specific nitrosamines in tobacco. The bacterial solution of *Bacillus curvatureis* Ni3 was sprayed onto the surface of cigar tobacco leaves. After a period of fermentation, tests showed that, compared to the control group, the TSNAs in the cigar tobacco leaves sprayed with *Bacillus curvatureis* Ni3 were significantly reduced. The specific implementation steps are as follows: Bacillus curvatureis Ni3 was inoculated onto LB agar plates and activated for 12 h. The activated Bacillus curvatureis Ni3 was then inoculated into liquid LB medium and fermented at 37°C and 180 rpm for 12 h in a shaker to prepare the fermentation broth. The fermentation broth was centrifuged at 12000 rpm for 5 min, the supernatant was removed, and the bacterial cells were collected. The collected bacterial cells were resuspended, and the bacterial concentration was determined using a spectrophotometer. The bacterial concentration was then diluted to 10⁻⁶. 8 cfu / mL is the fermentation agent of Bacillus curvatureis Ni3. A certain amount of two types of cigar tobacco leaves with high TSNA content were weighed and cut into Erlenmeyer flasks using sterile scissors as the fermentation medium for cigar tobacco leaves. The total moisture content of the cigar tobacco leaves was kept at 60%. A certain amount of the inoculum was added and evenly sprayed onto the tobacco leaves. Fermentation was carried out at 37°C and 75% RH for 72 hours.

[0043] After fermentation, cigar tobacco leaves were taken, dried at 60℃ for 3 hours, ground, passed through a 60-mesh sieve, and samples were taken. The contents of NNN, NNK, NAT, and NAB in the fermented cigar tobacco leaves were detected by YQ / T 29—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 Bacillus flexuralis Ni3 on TSNAs in different types of cigar tobacco leaves with high TSNA content was analyzed.

[0044] The effects of Bacillus curvatureis Ni3 on various TSNAs components in different types of cigar tobacco leaves were analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), as shown in Table 4 below.

[0045] Table 4. Effects of Ni3 on different types of cigar tobacco leaves Note: CX-81 refers to the Chuxue No. 81 cigar variety; CX-84 refers to the Chuxue No. 84 cigar variety. CK represents the control group.

[0046] As shown in the table above, *Bacillus campylobacter* Ni3 significantly reduced the TSNAs in fermented high-TSNA cigar tobacco leaves CX-81 and CX-84, with total TSNA content degradation rates reaching 46% and 43%, respectively. The degradation rates of various TSNA components in the two cigar tobacco leaves are as follows: Figure 6 , Figure 7 As shown.

[0047] Example 3: A microbial preparation The microbial preparation of this embodiment includes microorganisms and excipients, wherein the microorganism is Bacillus curvatureis (… Bacillus flexus Ni3, accession number CCTCC NO: M 20221334. The excipients can be those commonly used in this field.

[0048] In summary, the strain Ni3 described in this invention can efficiently reduce nicotine content and has a high degradation capacity for various components of TSNAs, including NNN, NNK, NAT, and NAB. Furthermore, it has demonstrated good degradation performance in practical applications with cigars. This bacterium has great application potential in reducing harmful substances in cigar tobacco leaves and their products, and can lay the foundation for subsequent optimization of microbial fermentation processes and improvement of cigar fermentation.

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas. It is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A Bacillus flexus Ni3, characterized in that: Bacillus flexus Ni3 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M20221334.

2. The application of Bacillus flexus as described in claim 1 in the degradation of tobacco-specific nitrosamines, characterized in that: The tobacco-specific nitrosamines mentioned are N-nitrosodemethylnicotine, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone, N-nitrosopseudoesequine, and N-nitrosoneonicotinine.

3. The application of Bacillus flexus according to claim 2 in the degradation of tobacco-specific nitrosamines, characterized in that: The tobacco mentioned is cigar tobacco.

4. A microbial preparation, characterized in that: It contains Bacillus flexus Ni3 as described in claim 1.

5. The application of the microbial preparation as described in claim 4 in the degradation of tobacco-specific nitrosamines in tobacco, characterized in that: The tobacco-specific nitrosamines mentioned are N-nitrosodemethylnicotine, 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone, N-nitrosopseudoesequine, and N-nitrosoneonicotinine.

6. An application of Bacillus flexus as described in claim 1 in the degradation of nicotine.

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