Pseudomonas and its application in degrading nicotine
Fermentation of cigar tobacco leaves with Pseudomonas J40 solved the problem of high nicotine content in tobacco, resulting in a significant improvement in tobacco quality, sensory quality, and industrial usability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEFEI UNIV OF TECH
- Filing Date
- 2026-04-24
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies are insufficient to effectively degrade the high nicotine content in tobacco, resulting in poor tobacco leaf quality and affecting the sensory quality and industrial usability of tobacco products.
Fermentation with Pseudomonas xanthophyll J40 was carried out through strain activation, seed liquid preparation, and fermentation broth preparation. The resulting product was then applied to cigar tobacco leaves to degrade nicotine content and improve tobacco quality.
It significantly reduces the nicotine content in cigar tobacco leaves, improves the quality of the tobacco leaves, produces a smooth and delicate smoke with reduced irritation, a clean aftertaste, and enhanced sensory quality.
Smart Images
Figure CN122104535A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a pseudoxanthomonas bacterium and its application in the degradation of nicotine. Background Technology
[0002] Nicotine is a major alkaloid in tobacco, accounting for over 93% of the total alkaloid content. It is not only an inducer of smoking addiction but also a highly toxic and non-degradable environmental pollutant. Excessive intake can suppress the central nervous system, leading to respiratory arrest and cardiac arrest. Due to the high incidence of smoking addiction caused by nicotine, tobacco and tobacco products have always been a focus of public concern.
[0003] Nicotine is a key indicator for evaluating tobacco leaf quality. High-quality filler tobacco not only requires certain sensory qualities but also a balanced ratio of chemical components. Compared to high-quality imported tobacco, the chemical composition of Chinese tobacco leaves is less balanced, especially in the upper filler leaves. This is mainly manifested in higher levels of nicotine and nitrogenous compounds, and key chemical indicators such as the sugar-to-nicotine ratio and sugar-to-nitrogen ratio failing to meet the standards for high-quality cigar tobacco. Sensory quality is characterized by a strong, irritating flavor, heavy off-flavors, and a rough smoke, significantly reducing the industrial usability of the tobacco and increasing the difficulty of blending.
[0004] In summary, how to reduce nicotine in tobacco leaves, improve the quality of low-quality tobacco leaves, and thus enhance their usability has been one of the most pressing problems to be solved in this field for many years. Summary of the Invention
[0005] The purpose of this invention is to provide a pseudoxanthomonas bacterium and its application in the degradation of nicotine.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A type of Xanthomonas, specifically Xanthomonas J40, is classified and named as follows: Pseudoxanthomonas sp., deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 20, 2025, with accession number CGMCC NO.36700.
[0007] The method for culturing Xanthomonas pseudoepiploicum according to the present invention includes the following steps: (1) Culture medium preparation; (2) Activation of strain: Pseudomonas xanthozogenes J40 was inoculated into LB solid medium and cultured at 37°C for 24 h until single colonies grew on the plate; (3) Seed culture preparation: Pick the single colony obtained in step (2) and inoculate it into LB liquid medium. Incubate at 37°C and shake at 150-170 rpm for 24 h. (4) Preparation of fermentation broth: The volume of the shake flask is 10~20% v / v. The seed liquid obtained in step (3) is inoculated into LB liquid medium at 2~5% v / v and cultured.
[0008] In step (1), the culture medium is LB solid medium and LB liquid medium. The LB solid medium is prepared by using 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar powder, 1000 mL distilled water, pH 7.0, and autoclaving at 121°C for 15 min. The LB liquid medium is prepared by using 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL distilled water, pH 7.0, and autoclaving at 121°C for 15 min.
[0009] In step (4), the culture temperature is 30~35℃, the shaking speed is 150~220rpm, and the culture time is 24~48h.
[0010] The present invention also provides the application of the above-mentioned *Xanthomonas aeruginosa* in the degradation of free nicotine.
[0011] The present invention also provides the application of the above-mentioned *Xanthomonas aeruginosa* in the fermentation and degradation of nicotine in cigar tobacco leaves.
[0012] Furthermore, this includes the following steps: (1) Pseudomonas xanthozogenes J40 was cultured to obtain fermentation broth and cell bodies; (2) Dilute the obtained fermentation liquid and bacteria with sterile water, and add the bacterial liquid at 20% of the weight of the tobacco leaves to cigar tobacco leaves with a water content of 20% for fermentation.
[0013] In step (2), the concentration of the diluted bacterial solution reaches 10. 9 bacteria / mL.
[0014] In step (2), the fermentation temperature is 30°C and the fermentation time is 7 days.
[0015] Compared with the prior art, the outstanding effect of the present invention is as follows: (1) In this invention, a strain of Xanthomonas pseudoxanthomonas J40 was screened and isolated. The fermentation broth of Xanthomonas pseudoxanthomonas J40 strain was obtained through strain activation, seed liquid preparation and fermentation broth preparation. It was then applied to the fermentation of cigar tobacco leaves, which can effectively reduce the nicotine content of cigar tobacco leaves and improve the quality of cigar tobacco raw materials, especially domestic cigar tobacco leaves. The process is simple and the reaction conditions are mild. After fermentation, the strength and impurities of domestic cigar tobacco leaves are significantly reduced, the smoke is soft and delicate, the irritation is reduced, and the aftertaste is clean.
[0016] (2) The method for degrading nicotine in this invention is simple, practical and effective, and can be promoted and applied in industry.
[0017] The following description, in conjunction with the accompanying drawings and specific embodiments, further illustrates the *Pseudomonas aeruginosa* of the present invention and its application in the degradation of nicotine.
[0018] Biological Preservation
[0019] Xanthomonas pseudoepiplos J40, classified and named Pseudoxanthomonas sp., deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on November 20, 2025, with accession number CGMCC NO. 36700. Attached Figure Description
[0020] Figure 1 This is a colony diagram of Xanthomonas pseudoxanthomonas J40.
[0021] Figure 2 This is a phylogenetic tree of Xanthomonas pseudoxanthomonas J40.
[0022] Figure 3 This is a graph showing the degradation curve of free nicotine by Pseudomonas J40.
[0023] Figure 4 for Staphylococcus Colony diagram of sp. Y3.
[0024] Figure 5 for Leifsonia shinshuensis Colony diagram of Y1.
[0025] Figure 6 for Chryseobacterium geocarposphaerae Colony diagram of J3. Detailed Implementation
[0026] Example 1: Isolation and identification of Xanthomonas pseudoepiploiculatus J40 (1) Test samples: The soil samples used were tobacco-growing soils, provided by the local tobacco company. Sampling location: Guangcun Town, Danzhou, Hainan, 109.3°E, 19.5°N, altitude 21.6 m.
[0027] (2) Preparation of culture medium: 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 pH to 7.0-7.5, and bring volume to 1000 mL.
[0028] Nicotine stock solution: Weigh 1 g of nicotine and dissolve it in 20 mL of sterile water. Filter the solution through a 0.22 μm filter membrane and store it in the refrigerator away from light.
[0029] Nicotine-rich medium: Take 0.3 mL of nicotine stock solution and bring the volume up to 50 mL with inorganic salt medium.
[0030] Nicotine selective medium: Add 2 g of agar powder to 100 mL of inorganic salt medium, autoclave at 121℃ for 15 min, and then add 0.2 mL of nicotine stock solution when the temperature is cooled to about 50℃.
[0031] (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 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 a 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 ( Figure 1 (As shown). After purification, the glycerol and bacterial solution were stored in a screw-top tube at a volume ratio of 1:1 and kept at -80°C.
[0032] (4) Classification and identification of strain J40: The obtained strain was subjected to 16S rRNA gene sequencing and its phylogenetic tree was constructed. Figure 2 Based on its physiological and biochemical characteristics, the bacterium was identified as belonging to *Xanthomonas pseudoepithecus*. Pseudoxanthomonas The 16S rRNA sequence of strain J40 is shown in SEQ ID NO:1.
[0033] Example 2: Application of J40 in the degradation of free nicotine (1) Xanthomonas pseudoepiploiculatus J40 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).
[0034] (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℃.
[0035] (3) The nicotine content in the sample was detected by LC-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.
[0036] (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 .
[0037] (5) After 12 h of degradation, the nicotine concentration in group J40 decreased significantly, and after 48 h, the degradation rate of nicotine by strain J40 reached a maximum of 96.9%. This indicates that strain J40 isolated and identified in this invention has a degradation effect on free nicotine.
[0038] Example 3: Application of J40 in the degradation of nicotine in cigar tobacco leaves (1) Xanthomonas pseudoepiploiculatus J40 was cultured to obtain fermentation broth. 10 mL of the fermentation broth was diluted to 10 mL with sterile distilled water. 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 Yunxue No. 1 cigar tobacco leaves with a moisture content of 20%. Sterile distilled water without fermentation liquid was set up as a blank control group (CK2).
[0039] (2) The experimental group and the blank control group (CK2) were fermented for 7 days at 30℃ 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%.
[0040] (3) The changes in chemical composition of fermented cigar tobacco leaves were determined by a flow analyzer. The results are shown in Table 1. Nicotine content decreased by 58.72%, and total nitrogen content decreased by 13.23%.
[0041] Table 1 Chemical composition of fermented cigar tobacco leaves
[0042] (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.
[0043] Table 2 Sensory Quality Evaluation Criteria for Cigar Tobacco Leaves
[0044] Note: In Table 2, the values 0 to 9 represent the scores used for evaluation. The concentration column is out of 5 points, and the other columns are out of 9 points.
[0045] Table 3 Sensory Quality Scores of Fermented Cigar Tobacco Leaves
[0046] To highlight the beneficial effects of the present invention, the following comparative experiments are provided.
[0047] Comparative Example 1 Compared with Example 3, the difference is that commercially available Pseudomonas bacteria were used for large-scale culture in the first step to obtain the fermentation broth.
[0048] Comparative Example 2 Compared with Example 3, the difference is that: in the first step, when J40 was isolated using Example 1, a nicotine-degrading bacterium was simultaneously screened. Staphylococcus sp. Y3, its colonies are as follows Figure 4 As shown, the culture was expanded to obtain the fermentation broth.
[0049] Comparative Example 3 Compared with Example 3, the difference is that: in the first step, when J40 was isolated in Example 1, a nicotine-degrading bacterium was simultaneously screened. Leifsonia shinshuensis Y1, its colonies are as follows Figure 5 As shown, the culture was expanded to obtain the fermentation broth.
[0050] Comparative Example 4 Compared with Example 3, the difference is that: in the first step, when J40 was isolated in Example 1, a nicotine-degrading bacterium was simultaneously screened. Chryseobacterium geocarposphaerae J3, its colonies are as follows Figure 6 As shown, the culture was expanded to obtain the fermentation broth.
[0051] The cigar tobacco leaves obtained in Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 were subjected to chemical composition analysis and sensory quality evaluation. The results of chemical composition analysis are shown in Table 1, and the sensory quality scores of the cigar tobacco leaves are shown in Table 3. The results indicate that after fermentation with J40, the nicotine content decreased by 58.72%, and the total nitrogen content decreased by 13.23%. In contrast, the nicotine content of tobacco leaves fermented with Pseudomonas aeruginosa decreased by only 37.21%, and the total nitrogen content decreased by 15.63%. The nicotine degradation rate of group Y3 was 41.8%, and the total nitrogen content decreased by 12.7%. The nicotine degradation rate of Y1 was 23.1%. J3 showed poor degradation effects on nicotine and nitrogen in the tobacco leaves, with the nicotine and total nitrogen content increasing rather than decreasing after fermentation.
[0052] This demonstrates that *Pseudomonas aeruginosa* J40 has a higher utilization rate and more thorough degradation effect on nicotine in tobacco leaves. Professional tasting results show that cigar tobacco treated with J40 is smoother and more delicate, with reduced irritation and a clean aftertaste. Table 3 shows that the J40 group had the lowest concentration score and the best effect in removing impurities; it also scored highest in terms of sweetness and smoothness.
[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A type of *Xanthomonas*, characterized in that: It is *Xanthomonas pseudoepithecus* J40, classified and named as follows: Pseudoxanthomonas sp., deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 20, 2025, with accession number CGMCC NO.36700.
2. The method for culturing *Xanthomonas pseudoepiplo* according to claim 1, characterized in that, Includes the following steps: (1) Culture medium preparation; (2) Activation of strain: Pseudomonas xanthozogenes J40 was inoculated into LB solid medium and cultured at 37°C for 24 h until single colonies grew on the plate; (3) Seed culture preparation: Pick the single colony obtained in step (2) and inoculate it into LB liquid medium. Incubate at 37°C and shake at 150-170 rpm for 24 h. (4) Preparation of fermentation broth: The volume of the shake flask is 10~20% v / v. The seed liquid obtained in step (3) is inoculated into LB liquid medium at 2~5% v / v and cultured.
3. The method for culturing *Xanthomonas pseudoepiplo* according to claim 2, characterized in that: The culture medium in step (1) is LB solid medium and LB liquid medium.
4. The method for culturing *Xanthomonas pseudoepiplo* according to claim 3, characterized in that: The LB solid culture medium is prepared by using 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar powder, 1000 mL distilled water, pH 7.0, and autoclaving at 121℃ for 15 min. The LB liquid culture medium is prepared by adding 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, and 1000 mL of distilled water at pH 7.0, and then autoclaving at 121°C for 15 min.
5. The method for culturing *Xanthomonas pseudoepiplo* according to claim 2, characterized in that: In step (4), the culture temperature is 30~35℃, the shaking speed is 150~220rpm, and the culture time is 24~48h.
6. The use of the *Xanthomonas pseudoepiploicum* as described in claim 1 in the degradation of free nicotine.
7. The application of *Xanthomonas pseudoepiplo* as described in claim 1 in the fermentation and degradation of nicotine in cigar tobacco leaves.
8. The application of *Xanthomonas pseudoepiplo* according to claim 7 in the fermentation and degradation of nicotine in cigar tobacco leaves, characterized in that: Includes the following steps: (1) Pseudomonas xanthozogenes J40 was cultured to obtain fermentation broth and cell bodies; (2) Dilute the obtained fermentation liquid and bacteria with sterile water, and add the bacterial liquid at 20% of the weight of the tobacco leaves to cigar tobacco leaves with a water content of 20% for fermentation.
9. The application of *Xanthomonas pseudoepiplo* according to claim 8 in the fermentation and degradation of nicotine in cigar tobacco leaves, characterized in that: In step (2), the concentration of the diluted bacterial solution reaches 10. 9 bacteria / mL.
10. The application of *Xanthomonas pseudoepiplo* according to claim 9 in the fermentation and degradation of nicotine in cigar tobacco leaves, characterized in that: In step (2), the fermentation temperature is 30℃ and the fermentation time is 7 days.