Staphylococcus warwick and its application in nicotine degradation

Abstract

This invention discloses a Staphylococcus warwickii strain and its application in the degradation of nicotine. The Staphylococcus warwickii strain of this invention is classified and named *Staphylococcus warwickii*. Staphylococcus warneri The specimen has been deposited with the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO. 36699, with a deposit date of November 20, 2025. This invention, *Staphylococcus warwickii*, applied to the fermentation of cigar tobacco leaves, can effectively reduce the nicotine content of cigar tobacco leaves and improve their quality.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Staphylococcus warwick and its application in the degradation of nicotine. Background Technology

[0002] Nicotine is the main alkaloid in plants of the Solanaceae family and the core active ingredient in tobacco, accounting for over 94% of the total alkaloid content. It is widely distributed throughout the leaves and roots of the tobacco plant, with the highest concentration in tobacco leaves. Nicotine is a highly toxic heterocyclic compound composed of a pyridine ring and a hydrogenated pyrrole ring. It is not only a key factor in smoking addiction but also a significant carcinogen. Long-term intake can cause serious damage to the respiratory, reproductive, and cardiovascular systems. Besides its harm to human health, nicotine also causes severe environmental pollution. Waste materials such as tobacco dust and stems generated during tobacco production and processing, smoke released from tobacco combustion, and the widespread use of neonicotinoid pesticides all contribute to nicotine entering the natural environment in various forms, polluting soil, water, and air. Therefore, reducing the nicotine content in tobacco and related environments is of significant practical importance and application value for human health protection and ecological environment governance.

[0003] Currently, nicotine degradation technologies are mainly divided into three categories: physical methods, chemical methods, and biological methods. However, physical and chemical methods suffer from drawbacks such as low degradation efficiency and complex equipment. In contrast, microbial methods, relying on various microorganisms and enzymes capable of degrading nicotine, have advantages such as lower cost, higher efficiency, and stronger environmental friendliness, and have become a research hotspot and mainstream development direction in the field of nicotine degradation. Currently, various microorganisms with nicotine degradation capabilities have been screened and identified both domestically and internationally, mainly including *Pseudomonas*, *Arthrobacter*, *Ailuropoda*, and *Enterobacter*. These strains are mostly isolated from tobacco planting soil, tobacco waste, or tobacco plants. Some strains have shown high nicotine degradation efficiency under laboratory conditions, but still have many shortcomings in practical applications. Summary of the Invention

[0004] Currently, the nicotine content of domestically produced cigar tobacco leaves is generally high, resulting in a strong, unpleasant aroma and limited industrial usability. To address this problem, this invention provides *Staphylococcus warwickii* and its application in nicotine degradation. The *Staphylococcus warwickii* provided by this invention, when applied to the fermentation of cigar tobacco leaves, can effectively reduce the nicotine content and improve the quality of the tobacco leaves.

[0005] This invention relates to Staphylococcus warwick, which is classified and named Staphylococcus warwick. Staphylococcus warneri The specimen, abbreviated as Staphylococcus warwick Y4, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 36699 and deposit date of November 20, 2025.

[0006] The present invention relates to the application of Staphylococcus warwick in the degradation of nicotine.

[0007] Furthermore, the present invention relates to the application of Staphylococcus warwick in the degradation of nicotine in cigar tobacco leaves.

[0008] The specific steps include:

[0009] Step 1: Activation of the strain

[0010] The *Staphylococcus wartii* Y4 was inoculated into LB solid medium and incubated at 37°C for 24 h until single colonies grew on the plates.

[0011] Step 2: Seed liquid preparation

[0012] Pick a single colony obtained from step 1 and inoculate it into LB liquid medium. Incubate at 37°C with shaking at 150-170 rpm for 24 h.

[0013] Step 3: Fermentation broth preparation

[0014] The volume of the shake flask is 10-20% (v / v). The seed culture obtained in step 2 is inoculated into LB liquid medium at 2-5% (v / v). The culture temperature is 30-35℃, the shaker speed is 150-220 rpm, and the culture time is 24-48 h.

[0015] Step 4: Nicotine Degradation

[0016] The fermentation broth obtained in step 3 was diluted with sterile water until the bacterial concentration reached 10. 9 One bacteria per mL: The diluted bacterial solution is added to cigar tobacco leaves for fermentation to reduce the nicotine content of the cigar tobacco leaves and improve the quality of the tobacco leaves.

[0017] The moisture content of the cigar tobacco leaves is 15%.

[0018] The added mass of the diluted bacterial solution is 20% of the mass of the cigar tobacco leaves.

[0019] The fermentation temperature is 35℃ and the fermentation time is 7 days.

[0020] The LB solid culture medium consists of the following components: 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar powder, 1000 mL distilled water, pH 7.0, and is autoclaved at 121°C for 15 min.

[0021] The LB liquid culture medium consists of: 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL distilled water, pH 7.0, and is autoclaved at 121°C for 15 min.

[0022] The beneficial effects of this invention are reflected in:

[0023] The method of this invention improves cigar tobacco raw materials, especially domestic cigar tobacco. The process is simple and the reaction conditions are mild. After fermentation, the strength and impurities of domestic cigar tobacco are significantly reduced, the smoke is smooth and delicate, the irritation is reduced, and the aftertaste is clean. This method is simple, practical, and effective, and can be widely applied in industry. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this embodiment. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a colony diagram of the strain of this invention.

[0026] Figure 2 This is a graph showing the degradation curve of free nicotine by the strain of this invention.

[0027] Figure 3 The results are the 16S rDNA sequencing results of the strain of this invention and the comparison results with the NCBI database. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0029] Example 1: Isolation and identification of Staphylococcus warwick Y4

[0030] (1) Test samples: The soil samples used were provided by the local tobacco company. The sampling location was Shigu Town, Shifang City, Deyang, Sichuan Province, 104.1°E, 31.2°N, altitude 594.3 m.

[0031] (2) Preparation of culture media: Inorganic salt culture medium: 1.5 g K2HPO4, 0.5 g KH2PO4, 0.2 g MgSO4, 1.0 g NH4SO4, 1.0 g NaCl, adjust the pH to between 7.0 and 7.5, and bring the volume to 1000 mL. Nicotine stock solution: Weigh 1 g of nicotine and dissolve it in 20 mL of sterile water, filter it through a 0.22 μm filter membrane, and store it in the dark under cold conditions. Nicotine-rich culture medium: Take 0.3 mL of nicotine stock solution and bring the volume to 50 mL with inorganic salt culture medium. Nicotine selective culture medium: Add 2 g of agar powder to 100 mL of inorganic salt culture medium, autoclave at 121℃ for 15 min, cool to about 50℃, and add 0.2 mL of nicotine stock solution.

[0032] (3) Bacterial cell isolation and purification: 2 g of ground and sieved soil sample was mixed with 50 mL of sterile water and cultured in a shaker at 37℃ and 150 r / min for 24 h to obtain an initial bacterial suspension. The bacterial suspension was centrifuged at 1500 rpm for 5 min, and the supernatant was collected. 2 mL of the supernatant was added to 50 mL of nicotine enrichment medium and cultured in a shaker at 37℃ and 150 r / min for 48-96 h. 10 μL of bacterial suspension was transferred to nicotine selective medium and spread evenly. The plates were then incubated at 37℃ for 48-72 h. After colonies grew on the plates, single colonies of different morphologies were picked and isolated by repeated streaking to obtain single colonies. After purification, the bacterial suspension was stored in a screw tube at -80℃ using a glycerol:bacterial suspension ratio of 1:1.

[0033] (4) Classification and identification of strain Y4: The obtained strain was subjected to 16S rRNA gene sequencing and its phylogenetic tree was constructed. Based on its physiological and biochemical characteristics, the applicant identified the strain as belonging to Staphylococcus warwickii ( Staphylococcus warneri This strain was sent to the China General Microbiological Culture Collection Center (CGMCC) on November 20, 2025, with accession number CGMCC NO. 36699 and classification name: *Staphylococcus warwickii*. Staphylococcus warneri .

[0034] Example 2: Application of Y4 in the degradation of free nicotine

[0035] (1) Staphylococcus warwick Y4 was cultured to obtain fermentation broth. The fermentation broth was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the broth was resuspended in sterile physiological saline. The centrifugation was repeated three times. The OD of the bacterial culture was adjusted with sterile physiological saline. 600 =0.8. Take 5 mL of bacterial culture and add 0.1 g of nicotine. Set up sterile physiological saline without bacterial culture as a blank control group (CK1).

[0036] (2) The experimental group and the blank control group (CK1) were cultured in a shaker at 37℃ and 150 r / min. Samples were taken every 12 h and stored at -80℃.

[0037] (3) The nicotine content in the sample was detected by UPLC-MS. The sample was thawed on ice and placed in the Starlid™ automated workstation for metabolite extraction; 100 μL of sample and 400 μL of extraction solution (methanol:acetonitrile = 1:1 (v / v), containing isotope internal standard) were transferred to a 96-well protein precipitation plate; the plate was shaken at 750 rpm for 5 min and allowed to stand for 5 min; the plate was filtered and the filtrate was collected.

[0038] (4) The target compounds were separated chromatographically using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph with a Phenomenex Kinetex C18 (2.1 mm × 50 mm, 2.6 μm) column. Phase A of the liquid chromatography was aqueous, containing 0.01% acetic acid, and phase B was isopropanol:acetonitrile (1:1, v / v). Sample tray temperature: 4℃, injection volume: 2 μL. The detection results are as follows: Figure 3 .

[0039] (5) After 12 h of degradation, the nicotine concentration in group Y4 decreased significantly, and after 48 h, the degradation rate of nicotine by strain Y4 reached a maximum of 81.1%. This indicates that strain Y4 isolated and identified in this invention has a degradation effect on free nicotine.

[0040] Example 3: Application of Y4 in the degradation of nicotine in cigar tobacco leaves

[0041] The specific steps are as follows:

[0042] (1) Staphylococcus warwick Y4 was cultured to obtain fermentation broth. 10 mL of the fermentation broth was diluted with sterile distilled water to a final concentration of 10 mL. 9 The bacterial culture was applied at a concentration of 100 microorganisms per mL, and 20% of the tobacco leaf weight was evenly sprayed onto the upper leaves of Yunxue No. 1 tobacco with a moisture content of 15%. Sterile distilled water without fermentation liquid was set up as a blank control group (CK2).

[0043] (2) The experimental group and the blank control group (CK2) were fermented for 7 days at 35℃ and 70% relative humidity. After fermentation, the tobacco leaves were spread out in an environment at 25℃ and 60% relative humidity to balance the moisture to 15%.

[0044] (3) The changes in chemical composition of fermented cigar tobacco leaves were determined by a flow analyzer. The results are shown in Table 1.

[0045] (4) The fermented cigar tobacco leaves were rolled into single-material tobaccos with a length of 70 mm and a diameter of 12 mm. The samples were then dried in a vacuum drying oven at 30°C for 3 h. The samples were evaluated by 5 professionals based on 7 indicators: concentration, aroma quality, aroma quantity, irritation, off-flavors, smoothness, and sweetness. The evaluation criteria are shown in Table 2, and the sensory quality scores of the cigar tobacco leaves are shown in Table 3.

[0046]

[0047]

[0048]

[0049] Table 2 shows that the nicotine and total nitrogen content of cigar tobacco leaves fermented with Staphylococcus wartii Y4 decreased compared with the blank control group (CK2). According to professional evaluation, the fermented cigar tobacco leaves showed a significant reduction in strength and impurities, resulting in a smoother, more delicate smoke with reduced irritation and a cleaner aftertaste.

[0050] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims. It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. Furthermore, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, they should also be considered as the content disclosed in the present invention.

Claims

1. A Staphylococcus warwickii strain, characterized in that: The strain of Staphylococcus wartii is classified as Staphylococcus wartii. Staphylococcus warneri It has been deposited with the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO. 36699, with a deposit date of November 20, 2025.

2. The application of Staphylococcus warwick as described in claim 1 in the degradation of nicotine.

3. The use of Staphylococcus warwick as described in claim 1 in the degradation of nicotine in cigar tobacco leaves.

4. The application according to claim 3, characterized in that... Includes the following steps: Step 1: Activation of the strain The *Staphylococcus wartii* was inoculated into LB solid medium and incubated at 37°C until single colonies grew on the plates. Step 2: Seed liquid preparation Select a single colony obtained from step 1 and inoculate it into LB liquid medium. Incubate at 37°C with shaking at 150-170 rpm for 24 h. Step 3: Fermentation broth preparation The volume of liquid in the shake flask is 10-20%, and the seed liquid obtained in step 2 is inoculated into LB liquid medium at a rate of 2-5% for fermentation culture. Step 4: Nicotine Degradation The fermentation liquid obtained in step 3 is diluted with sterile water, and the diluted bacterial liquid is evenly sprayed onto the surface of cigar tobacco leaves for fermentation treatment to reduce the nicotine content of cigar tobacco leaves and improve the quality of tobacco leaves.

5. The application according to claim 4, characterized in that: In step 3, the culture temperature is 30~35℃, the shaking speed is 150~220rpm, and the culture time is 24~48h.

6. The application according to claim 4, characterized in that: In step 4, the fermentation broth obtained in step 3 is diluted with sterile water to a concentration of 10. 9 bacteria / mL.

7. The application according to claim 4, characterized in that: The moisture content of the cigar tobacco leaves is 15%.

8. The application according to claim 4, characterized in that: The added mass of the diluted bacterial solution is 20% of the mass of the cigar tobacco leaves.

9. The application according to claim 4, characterized in that: The fermentation temperature is 35℃ and the fermentation time is 7 days.

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