A strain of Rhodococcus fannings and its application in the efficient degradation of nicotine.

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Rhodococcus qingshengii* and its application in the efficient degradation of nicotine. Specifically, this invention screened a bacterium with stable nicotine degradation capabilities from the rhizosphere environment of tobacco soil. Identified as *Rhodococcus qingshengii*, it was named Cas430 (*Rhodococcus qingshengii*). This bacterium grows rapidly and has strong environmental adaptability, utilizing nicotine as its sole carbon and nitrogen source for growth. It exhibits highly efficient degradation capabilities; after curing, the nicotine content of the tobacco leaves sprayed with this bacterium is reduced, effectively improving the quality of tobacco products and significantly reducing the harm of smoking to human health. Simultaneously, this strain can also be used to degrade nicotine-containing waste generated during tobacco product manufacturing or to treat nicotine in tobacco plantations and water sources, thereby improving the environment and possessing significant economic, social, and ecological benefits.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Rhodococcus fanningii and its application in the efficient degradation of nicotine. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Nicotine (C 10 H 14 Nicotine (N2) is a naturally occurring pyridine alkaloid found in tobacco plants. As a major addictive component of tobacco products, its harmfulness has attracted widespread attention. Nicotine not only directly harms human health through smoking but also enters the environment through tobacco processing waste, discarded cigarette butts, and residues in tobacco field soil, causing persistent pollution. From a health perspective, nicotine can activate acetylcholine receptors in the central nervous system, leading to increased dopamine release and resulting in physiological and psychological dependence. Long-term nicotine intake significantly increases the risk of cardiovascular disease, respiratory diseases, and various cancers (such as lung cancer and oral cancer). Furthermore, nicotine can cross the placental barrier and affect fetal and infant development through breast milk, leading to neurobehavioral abnormalities and cognitive impairment. Simultaneously, due to its chemical stability, nicotine persists in soil and water bodies for extended periods. Nicotine in the environment inhibits soil microbial activity, interferes with plant root development, and accumulates in aquatic organisms through the food chain, ultimately threatening biodiversity and ecological security.

[0004] To address nicotine pollution, existing technologies are mainly divided into two categories: physicochemical methods and biological methods. Physicochemical methods include high-temperature incineration and acid / alkali treatment, adsorption of nicotine using activated carbon, zeolite, or polymeric materials, decomposition of nicotine under ultraviolet light using catalysts such as TiO2 and ZnO, and degradation of nicotine using strong oxidants such as ozone and persulfate. However, these methods suffer from high degradation costs, poor adaptability to complex substrates (such as soil), and a tendency to generate secondary pollution. Biodegradation has become a research hotspot due to its environmental friendliness and low cost. Currently reported nicotine-degrading bacteria include *Pseudomonas* and *Alcaligenes*. However, existing strains generally suffer from low degradation efficiency, high substrate dependence, and limited application scenarios. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a strain of *Rhodococcus qingshengii* and its application in the efficient degradation of nicotine. Specifically, this invention screened a bacterium with stable nicotine degradation capabilities from the rhizosphere environment of tobacco soil. This bacterium was identified as *Rhodococcus qingshengii* and named Cas430. This bacterium grows rapidly and exhibits strong environmental adaptability, capable of utilizing nicotine as its sole carbon and nitrogen source for growth, demonstrating highly efficient degradation capabilities and significant research value. Based on the above research findings, this invention is thus completed.

[0006] To achieve the above-mentioned technical objectives, the present invention relates to the following technical solutions:

[0007] In a first aspect, the present invention provides a strain of Rhodococcus qingshengii Cas430, which was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 32934.

[0008] A second aspect of the present invention provides a fermentation production method for Rhodococcus faecalis Cas430, the fermentation production method comprising: inoculating the Rhodococcus faecalis Cas430 into a fermentation medium for fermentation culture.

[0009] In this invention, no specific limitation is made to the fermentation production method; any conventional bacterial fermentation culture method can be used for cultivation.

[0010] The fermentation medium can be any common bacterial culture medium, such as NB liquid culture medium in one specific embodiment of the present invention.

[0011] A third aspect of the present invention provides a microbial inoculant containing at least the aforementioned Rhodococcus faecalis Cas430.

[0012] In this invention, the microbial agent may further include acceptable excipients in the agent.

[0013] In another specific embodiment of the present invention, the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers, and solvents. The present invention does not impose any special restrictions on the sources of acceptable excipients for the bacterial agent; commercially available products are generally sufficient.

[0014] The dispersant is an anionic dispersant and / or a nonionic dispersant, and may be selected from one or more of sodium lignosulfonate, sodium naphthalenesulfonate formaldehyde condensate, sodium methylene bisnaphthalenesulfonate, formaldehyde condensate sulfate, polycarboxylate, alkylphenol polyoxyethylene phosphate, and fatty acid polyoxyethylene ester.

[0015] The wetting agent may be selected from one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, soapberry powder, soapberry powder, tea seed cake powder, and splitting powder BX.

[0016] The disintegrant may be selected from one or more of bentonite, ammonium sulfate, aluminum chloride, urea, magnesium chloride, and glucose.

[0017] The binder may be selected from one or more of starch, diatomaceous earth, cyclodextrin, rosin, carboxymethyl cellulose, carboxyethyl cellulose, and carboxymethyl cellulose salts.

[0018] The defoamer may be selected from one or more of the following: C8-C20 fatty alcohols, C10-C20 saturated fatty acid compounds, epoxidized soybean oil, ethanol, silicone compounds, and organosilicon oil.

[0019] The antifreeze agent may be selected from one or more of sorbitol, ethylene glycol, polyethylene glycol, propylene glycol, glycerol, urea, and sodium chloride.

[0020] The thickener may be selected from one or more of gelatin, xanthan gum, polyethylene glycol, and polyvinyl alcohol.

[0021] The filler may be selected from one or more of the following: light calcium carbonate, diatomaceous earth, bentonite, attapulgite, and silica.

[0022] The solvent may be selected from water (preferably deionized water) or methyl oleate.

[0023] A fourth aspect of the present invention provides the application of the above-mentioned Rhodococcus faecalis Cas430 and / or the above-mentioned microbial agents in nicotine degradation.

[0024] A fifth aspect of the present invention provides the application of the above-mentioned Rhodococcus Cas430 and / or the above-mentioned microbial agents in the degradation of nicotine in tobacco products, tobacco industrial waste, tobacco plants, soil of tobacco growing areas and / or water sources of tobacco growing areas.

[0025] In this invention, the tobacco products include, but are not limited to, tobacco leaves, re-dried tobacco leaves, tobacco sheets, shredded tobacco, cigarettes, and cigars; the tobacco industry waste includes, but is not limited to, tobacco stems and tobacco dust.

[0026] In this invention, the tobacco shreds refer to shredded, powdered, or granular products made from tobacco leaves, re-dried tobacco leaves, and tobacco sheets.

[0027] A sixth aspect of the present invention provides a method for degrading nicotine, the method comprising the steps of treating tobacco products, tobacco plants, soil of tobacco growing areas and / or water sources of tobacco growing areas with the aforementioned Rhodococcus Cas430 and / or the aforementioned microbial agents.

[0028] A seventh aspect of the present invention provides the use of the above-mentioned Rhodococcus faecalis Cas430, microbial agents and / or methods in the preparation of cigarettes and / or e-liquids.

[0029] In this invention, the e-liquid can be e-liquid from electronic cigarettes and / or other non-combustible cigarette products.

[0030] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0031] The above-mentioned technical solution reports for the first time a highly efficient nicotine-degrading Rhodococcus fanningii strain. This strain has the ability to grow using nicotine as the sole carbon and nitrogen source. After 24 hours of cultivation on BMM medium containing 1 g / L nicotine, the nicotine content can be reduced by more than 80%, and nicotine can be completely degraded after 42 hours. It exhibits high nicotine degradation efficiency and preferentially uses nicotine as a metabolite when other carbon and nitrogen sources are available, demonstrating a strong nicotine metabolism capacity. Spraying the strain on tobacco plants for 12 hours reduced the nicotine content by approximately 20.34%. The nicotine content of the sprayed tobacco leaves was reduced after curing, thus effectively improving the quality of tobacco products and significantly reducing the harm of smoking to human health. Simultaneously, this strain can also be used to degrade nicotine-containing waste generated in tobacco product manufacturing, achieving comprehensive utilization of waste, or to treat nicotine in tobacco planting areas and water sources, thereby improving the environment. It has significant economic, social, and ecological benefits and therefore has broad application prospects. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 The colony morphology of strain Cas430 (Rhodococcus qingshengii) in the embodiments of the present invention is shown.

[0034] Figure 2 In this embodiment of the invention, the Neighbour-Joining method was used to construct a phylogenetic tree of Cas430 (Rhodococcus qingshengii) and other standard species of Rhodococcus.

[0035] Figure 3The nicotine degradation curve of strain Cas430 (Rhodococcus qingshengii) in this embodiment of the invention after being cultured in BMM medium containing 1 g / L nicotine is shown. The peak area is the nicotine absorption peak area detected by HPLC (black for 18 h, blue for 24 h, green for 42 h, and cyan for 48 h).

[0036] Figure 4 The growth rate of strain Cas430 (Rhodococcus qingshengii) in the embodiments of the present invention after culturing in BMM medium containing 1, 2, 3, 4, and 5 g / L nicotine at 16 h, 20 h, 24 h, 30 h, 40 h, and 48 h.

[0037] Figure 5 The peak areas of strain Cas430 (Rhodococcus qingshengii) in this embodiment of the invention were measured by liquid chromatography at 16h, 20h, 24h, 30h, 40h and 48h after culturing in BMM medium containing 1, 2, 3, 4 and 5 g / L nicotine. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0039] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0040] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0041] Example

[0042] 1. Materials and Methods

[0043] 1.1 Materials

[0044] Experimental sample: Soil sample from the root surface of tobacco plants in Hubei Province.

[0045] Required culture media: NA medium, NB liquid medium, BMM medium (KH2PO4, 3.6g; (NH4)2SO4, 2g; L-glutamic acid, 2.9g; glucose, 0.18g; FeSO4-7H2O, 0.5mg (pH=6.5)), LB medium.

[0046] Required sample: Nicotine (purity: HPLC ≥ 95%)

[0047] 1.2 Methods

[0048] 1.2.1 Isolation and culture of strains

[0049] Rhizosphere soil samples were collected from tobacco plants infected with bacterial wilt in Hubei Province in August 2022. After shaking off the soil adhering to the root surface, the root tissue samples were placed in sterilized deionized water, and the root surface soil was collected using an ultrasonic agitation method. Centrifuge tubes were placed in water and ultrasonically treated for 20 seconds each time, for a total of two treatments. Fine roots in the centrifuge tubes were removed and discarded using sterile forceps in a laminar flow hood. 1 mL of the soil suspension was added to 9 mL of sterilized deionized water, shaken thoroughly, and kept at 10 mL / min. -1 Similarly, prepare 10 times the bacterial culture. -2 10 -3 10 -4 10 -5 and 10 -6 A bacterial suspension diluted 10 times. Take samples with a concentration of 10... -3 10 -4 10 -5 and 10 -6 0.1 mL of each culture was used, with three replicates for each dilution. The cultures were spread onto NA medium using a disposable spreader and incubated at 28°C for 48 hours. Single colonies were selected based on growth characteristics such as color, size, elevation, transparency, firmness, and edge regularity. These single colonies were then streaked three times on NA medium to obtain pure cultures. The strain with the best growth was selected for identification and further investigation into its nicotine-degrading effect.

[0050] 1.2.2 Identification of strain species

[0051] ① Streak the strain onto fresh NA medium and incubate at 28°C for 24 hours. Use a 100 μL pipette tip to pick up the purified bacterial colony into a 2 mL centrifuge tube, add 100 μL of sterile water, boil in a water bath for 10 minutes, cool, centrifuge at 12000 rpm for 10 minutes, and collect the supernatant as the DNA of the strain.

[0052] ② Using genomic DNA as a template, PCR amplification was performed using universal bacterial primers 27F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492F (5′-AAGGAGGTGATCCAACCGCA-3′). The PCR reaction mixture (50 μL) consisted of 2 μL DNA template, 25 μL 2×Taq Plus Master Mix II (Dye Plus), 1.5 μL each of 27F and 1492R (10 μmol / L), and ddH2O to a final volume of 50 μL. PCR reaction conditions were: 95°C for 3 min; 95°C for 30 s, 58°C for 30 s, 72°C for 1.5 min, for 30 cycles; 72°C for 5 min. After PCR amplification products were detected by 1% agarose gel electrophoresis, they were sent to NIO Bio for sequencing. The sequencing splicing results were compared with BLAST similar sequences on NCBI, and multiple sequence homology analysis was performed using MEGA software. A phylogenetic tree was constructed, and the taxonomic position of the strain was finally determined based on the similarity and position in the phylogenetic tree.

[0053] 1.2.3 Preservation of microbial strains

[0054] The purified strain was streaked on a slant, and single colonies that grew after streaking were picked up with an inoculation loop and transferred to NB liquid medium. The culture was incubated at 28°C and 180 rpm until the exponential growth phase. The bacteria were then preserved in 50% glycerol aqueous solution, aliquoted into 2 mL cryovials, and stored at -80°C for later use. The culture was then sent to the China General Microbiological Culture Collection Center (CGMCC) with the preservation number CGMCCNo. 32934.

[0055] 1.2.4 Establishment of Nicotine Liquid Chromatography Standard Curve

[0056] Nicotine was prepared into seven concentration gradient standard solutions of 2 g / L, 0.5 g / L, 0.25 g / L, 0.125 g / L, 0.0625 g / L and 0.03125 g / L, and a nicotine concentration standard curve was established using high performance liquid chromatography.

[0057] The chromatographic column was an Agilent TC-C18; the mobile phase was 40% acetonitrile; the column temperature was 30℃; the flow rate was 0.6 mL / min; the injection volume was 20 μL; and the detection wavelength was 259 nm.

[0058] 1.2.5 Effect of fermentation time on the nicotine degradation efficiency of Cas430 (Rhodococcus qingshengii)

[0059] Cas430 (Rhodococcus qingshengii) was cultured in BMM medium containing 1 g / L nicotine. Samples were taken at 18 h, 24 h, 42 h, and 48 h, and the nicotine degradation rate was detected by liquid chromatography. 1 mL of fermentation broth was centrifuged at 15000 rpm for 2 min, the bacterial cells were discarded, and the supernatant was filtered through a 0.22 μm organic phase filter and then placed in a liquid chromatography vial for HPLC detection.

[0060] The chromatographic column was an Agilent TC-C18; the mobile phase was 40% acetonitrile; the column temperature was 30℃; the flow rate was 0.6 mL / min; the injection volume was 20 μL; and the detection wavelength was 259 nm.

[0061] 1.2.6 Effect of fermentation concentration on the nicotine degradation efficiency of Cas430 (Rhodococcus qingshengii)

[0062] Strains Cas430 (Rhodococcus qingshengii) were inoculated at a 1% inoculum into nicotine liquid medium with nicotine concentrations of 1, 2, 3, 4, and 5 g / L and cultured at 28°C and 200 rpm. Sampling was taken every 4 hours to measure bacterial growth and the degradation rate at different time points under different nicotine concentrations.

[0063] 1.2.7 Degradation of Nicotine on Tobacco Plants

[0064] 1) Preparation of bacterial culture

[0065] Cas430 was cultured overnight in LB liquid medium until OD reached. 600 =1.0, dilute the bacterial solution 10 times with deionized water for later use.

[0066] 2) Handling methods

[0067] Select tobacco plants with uniform growth after topping, spray the entire plant twice, and spray the control group with water. Three tobacco plants were used for each treatment. After spraying, four leaves from each plant were taken 12 hours later, placed in an envelope and dried in a 72-degree oven, and then ground into powder for later use.

[0068] 3) Detection methods

[0069] For each treatment, 10g of dried tobacco leaf samples were taken, and the nicotine content was detected using a Fourier transform near-infrared spectrometer.

[0070] 2. Experimental Results

[0071] 2.1.1 Morphological characteristics of the strain

[0072] Cas430 (Rhodococcus qingshengii) was inoculated onto NA medium and incubated at 28°C for 24 hours. Single colonies of the strain were pale yellow, slightly transparent, and had smooth edges. Figure 1 ).

[0073] 2.1.2 Identification of strain species

[0074] The nucleotide sequence shown in SEQ ID NO.1 was obtained by 16S rDNA sequencing. Comparison of the obtained sequence with the EZBioCloud database revealed that Cas430 (Rhodococcus qingshengii) had the highest similarity (99.78%) to Rhodococcus qingshengii JCM 15477. A phylogenetic tree was constructed by combining the 16S rDNA sequences of Cas430 with those of other standard Rhodococcus species. Figure 2 It was discovered that Cas430 and Rhodococcus fanqingsheng clustered together, and Cas430 was ultimately identified as Rhodococcus fanqingsheng.

[0075] 2.2 Effect of fermentation time on the nicotine degradation efficiency of Cas430 (Rhodococcus qingshengii)

[0076] Cas430 (Rhodococcus qingshengii) was cultured in BMM medium containing 1 g / L nicotine. Samples were taken at 18 h, 24 h, 42 h, and 48 h, and the nicotine degradation rate was detected by liquid chromatography. The results showed that the degradation rate of Cas430 was 52.49% at 18 h; 78.76% at 24 h; and at 42 h, the liquid chromatography showed that the nicotine was completely degraded. Figure 3 ).

[0077] 2.3 Effect of fermentation concentration on the nicotine degradation efficiency of Cas430 (Rhodococcus qingshengii)

[0078] Strains of Cas430 were inoculated at a rate of 1% into nicotine liquid culture media with nicotine concentrations of 1, 2, 3, 4, and 5 g / L, and cultured accordingly. OD 600 The results showed that the growth of strain Cas430 was not inhibited in nicotine liquid medium at concentrations of 1 and 2 g / L, while the growth was slower at concentrations of 3, 4, and 5 g / L compared to 1 and 2. Liquid phase results indicated that strain Cas430 degraded nicotine rapidly in nicotine liquid medium at concentrations of 1 and 2 g / L, with nicotine at 1 g / L being completely degraded within 48 hours, while the degradation rate of nicotine was very slow at concentrations of 3, 4, and 5 g / L.

[0079] 2.4 Degradation of Nicotine on Tobacco Plants

[0080] Analysis of the results of the treated tobacco leaf samples by Fourier transform near-infrared spectroscopy showed that spraying tobacco plants with a bacterial solution containing Cas430 and its fermentation products for 12 hours reduced the nicotine content of the tobacco plants by 20.34%.

[0081] In summary, this invention isolated a bacterium from the root surface of diseased tobacco plants and named it Cas430. 16S rRNA sequencing and phylogenetic tree construction confirmed that strain Cas430 is *Rhodococcus qingshengii*. Experimental results demonstrated that strain Cas430 has the ability to grow using nicotine as the sole carbon and nitrogen source. Cultured in BMM medium containing 0.1 g / L nicotine for 30 hours, it reduced the nicotine content by 95.16%. Even in the presence of other carbon and nitrogen sources, this bacterium still preferentially utilizes nicotine as a metabolite, exhibiting a strong nicotine metabolism capacity. Cultivation on BMM medium containing 1 g / L nicotine for 24 hours can reduce the nicotine content by more than 80%, and complete degradation of nicotine is achieved within 42 hours, demonstrating high nicotine degradation efficiency. When strain Cas430 was inoculated at a 1% inoculum into nicotine liquid medium with nicotine concentrations of 1, 2, 3, 4, and 5 g / L, the results showed that the growth of strain Cas430 was not inhibited in nicotine liquid medium at 1 and 2 g / L, and nicotine at 1 g / L was completely degraded within 48 hours. Compared to 1 and 2 g / L, the growth of strain Cas430 at the 3, 4, and 5 g / L concentrations was slower.

[0082] This bacterium also has a significant ability to degrade nicotine in tobacco plants. Spraying tobacco plants with a bacterial solution containing Cas430 (Rhodococcus qingshengii) and its fermentation products for 12 hours reduced the nicotine content of the tobacco plants by about 20.34%.

[0083] Cas430 16S rRNA gene sequence

[0084] TGCAGTCGAGCGGTAAGGCCTTTCGGGGTACACGAGCGGCGAACGGG

[0085] TGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGA

[0086] AACTGGGTCTAATACCGGATATGACCTCCTATCGCATGGTGGGTGGTGG

[0087] AAAGATTTATCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTG

[0088] GGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGT

[0089] GACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG

[0090] CAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGAC

[0091] GCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGG

[0092] GACGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTA

[0093] CGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAAT

[0094] TACTGGGCGTAAAGAGTTCGTAGGCGGTTTGTCGCGTCGTTTGTGAAA

[0095] ACCAGCAGCTCAACTGCTGGCTTGCAGGCGATACGGGCAGACTTGAGT

[0096] ACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGAT

[0097] ATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTG

[0098] ACGCTGAGGAACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTG

[0099] GTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTCCTTCCACG

[0100] GAATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACG

[0101] GCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG

[0102] GCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGG

[0103] TTTGACATATACCGGAAAGCTGCAGAGATGTGGCCCCCCTTGTGGTCG

[0104] GTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGG

[0105] GTTAAGTCCCGCAACGAGCGCAACCCCTATCTTATGTTGCCAGCACGTT

[0106] ATGGTGGGGACTCGTAAGAGACTGCCGGGGTCAACTCGGAGGAAGGT

[0107] GGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACA

[0108] TGCTACAATGGCCAGTACAGAGGGCTGCGAGACCGTGAGGTGGAGCG

[0109] AATCCCTTAAAGCTGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACC

[0110] CCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTG

[0111] AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTC

[0112] GGTAACACCCGAAGCCGGTGGCTTAACCCCTTGTGGGAGGGAGCCGT

[0113] CGAA(SEQ ID NO.1)

[0114] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A type of Rhodococcus faecalis (Fan Qingsheng) Rhodococcus qingshengii The application of Cas430 and / or microbial agents in nicotine degradation; the Rhodococcus fanqingsheng Cas430 was deposited at the China General Microbiological Culture Collection Center on December 5, 2024, with the biological accession number CGMCC No. 32934. The microbial agent contains at least Rhodococcus faecalis Cas430.

2. The application as described in claim 1, characterized in that, The microbial agent also includes acceptable excipients; the excipients are selected from one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents and thickeners.

3. The application of Rhodococcus faecalis Cas430 and / or the microbial agent described in claim 1 in the degradation of nicotine in tobacco products, tobacco industry waste, tobacco plants, soil of tobacco growing areas and / or water sources of tobacco growing areas.

4. The application as described in claim 3, characterized in that, The tobacco products include tobacco leaves, shredded tobacco, cigarettes, and cigars; the tobacco industry waste includes tobacco stems and tobacco dust.

5. A method for degrading nicotine, characterized in that, The method includes the steps of treating tobacco products, tobacco plants, tobacco growing soil and / or tobacco growing water sources using the Rhodococcus Cas430 of Fan Qingsheng in the application of claim 1 and / or the microbial agent.

6. The use of *Rhodococcus faecalis* Cas430 and / or the microbial agent described in claim 1 and / or the method described in claim 5 in the preparation of cigarettes and / or e-liquid.

7. The application as described in claim 6, characterized in that, The e-liquid is from non-combustible cigarette products.

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