Stenotrophomonas maltophilia YG-17 capable of efficiently degrading nicotine and application of stenotrophomonas maltophilia YG-17

Abstract

The invention relates to stenotrophomonas maltophilia YG-17 capable of efficiently degrading nicotine and application of the stenotrophomonas maltophilia YG-17, the classification name of the stenotrophomonas maltophilia YG-17 is Shenotrophomonas maltophilia YG-17, the preservation number of the stenotrophomonas maltophilia YG-17 is CCTCC (China Center For Type Culture Collection) NO: M 2025684, and the preservation date of the stenotrophomonas maltophilia YG-17 is April 2, 2025. The strain has excellent nicotine degradation capability, enriches nicotine degradation strain resources, has potential application value in the production process of tobacco products, and can reduce the health risk of the tobacco products to human bodies; the strain can also be applied to biodegradation of nicotine-containing tobacco waste generated in the tobacco industry, and cyclic utilization of waste resources is promoted; nicotine pollution in soil or waste water in a tobacco planting area can be treated in an environment-friendly mode, environmental protection is facilitated, and harm to the environment and ecology is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of strain development, and relates to a new strain for efficiently degrading nicotine and an application thereof, in particular to a Stenotrophomonas maltophilia YG-17 for efficiently degrading nicotine and an application thereof. Background Art

[0002] Alkaloids primarily include nicotine, nornicotine, anatabine, and anabasine, with nicotine accounting for over 94% of the total alkaloid content in tobacco. Nicotine distribution varies widely throughout the tobacco plant, with the highest concentrations in leaves, followed by roots, stems, flowers, and fruit. Nicotine content in tobacco seeds is generally very low.

[0003] Nicotine, commonly known as nicotine, is a highly toxic compound. It is a heterocyclic compound composed of two nitrogen heterocycles: a pyridine ring and a hydropyrrole ring, and is weakly alkaline overall. Nicotine is a key component of smoking addiction and also causes certain damage to the human body. Furthermore, since nicotine typically enters the natural environment in the form of smoke or sewage, its environmental pollution comes from multiple sources, including nicotine contamination in tobacco waste generated during tobacco production, nicotine pollution from tobacco combustion, and the widespread use of neonicotinoid pesticides. If tobacco waste rich in nicotine and its derivatives is not properly treated, it can endanger drinking water safety, reduce the number of terrestrial and aquatic organisms, and even damage ecosystems.

[0004] Currently, nicotine degradation primarily involves agricultural technology and physical and chemical approaches. Within the agricultural technology sector, nicotine content regulation is primarily achieved through a multi-faceted approach: first, conventional breeding to select low-nicotine germplasm resources; second, gene editing to precisely manipulate nicotine biosynthesis pathways; and third, the application of cultivation control strategies, such as optimizing nutrient supply and adjusting harvest timing. However, existing agricultural technology approaches suffer from limitations such as high genetic complexity, strong environmental dependence, and low economic viability. Furthermore, they struggle to balance nicotine control with plant health and industrial needs. Among physical methods, solvent extraction has achieved significant progress in nicotine separation efficiency, but its industrial application remains limited by bottlenecks such as high energy consumption, complex process parameter control, and high equipment maintenance costs. Chemical approaches to nicotine pollution control in water bodies primarily rely on oxidation technologies, including photocatalytic degradation, Fenton oxidation, and piezoelectric catalytic systems. However, these technologies generally suffer from technical limitations such as insufficient energy efficiency, low oxidant utilization, and the risk of secondary pollution. The development of green and efficient co-processing technologies is urgently needed.

[0005] Due to the specificity of microbial fermentation, its impact on other chemical components in tobacco leaves is very limited, and it does not degrade the intrinsic or external quality of the tobacco leaves. Therefore, nicotine degradation has become a hot topic in research. Currently, the most widely studied and applied nicotine-degrading microorganisms are Arthrobacter and Pseudomonas. Several strains with nicotine-degrading properties have also been isolated and identified from tobacco soil and leaves, such as Rhodococcus sp. Y22, Ensifer sp. N7, Acinetobacter sp. TW, and Ochrobactrum sp. SJY1.

[0006] While current microbial fermentation technology for nicotine degradation is environmentally friendly, practical applications still suffer from low nicotine degradation efficiency and a difficulty in achieving both high activity and strong environmental adaptability. Furthermore, despite progress in basic research, engineering applications within tobacco agricultural production systems, particularly in nicotine degradation in processing byproducts such as tobacco stalks and tobacco dust, have yet to achieve a technological breakthrough worthy of industrialization. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a new strain that can efficiently degrade nicotine and its application, in particular to provide a new strain of Stenotrophomonas maltophilia YG-17 that can efficiently degrade nicotine and its application.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a Stenotrophomonas maltophilia YG-17 that efficiently degrades nicotine. The classification name of the Stenotrophomonas maltophilia YG-17 is Stenotrophomonas maltophilia, the preservation number is CCTCC NO: M2025684, and the preservation date is April 2, 2025.

[0010] The present invention isolated and preserved a new strain of Stenotrophomonas maltophilia that is capable of efficiently degrading nicotine from the soil of tobacco plant roots. Sequencing analysis revealed that the 16S rDNA sequence of the strain is shown in SEQ ID No: 1. A nucleic acid sequence alignment of the sequenced sequence revealed that the strain is Stenotrophomonas maltophilia. The strain has excellent nicotine-degrading ability, which makes it potentially useful in tobacco product production, reducing the health risks of tobacco products to humans. It can also be used to biodegrade nicotine-containing tobacco waste generated by the tobacco industry, promoting the recycling of waste resources. It can also be used to environmentally friendly treat nicotine contamination in soil or wastewater in tobacco-growing areas, contributing to environmental protection and avoiding environmental and ecological harm. Furthermore, the strain can enrich the resource of nicotine-degrading strains and be used to screen for genes related to efficient nicotine degradation, providing a new gene source for transgenic tobacco and engineered strains, thereby improving the quality of tobacco products and reducing the harm of nicotine to humans and the environment.

[0011] In a second aspect, the present invention provides use of the Stenotrophomonas maltophilia YG-17 or a fermentation product thereof described in the first aspect in the preparation of a nicotine degrader.

[0012] In a third aspect, the present invention provides a preparation for degrading nicotine, wherein the strain in the preparation for degrading nicotine includes the Stenotrophomonas maltophilia YG-17 described in the first aspect.

[0013] Preferably, the viable count of Stenotrophomonas maltophilia YG-17 in the preparation is not less than 6×10 6 CFU / mL or 6×10 6 CFU / g, for example 6×10 6 CFU / g (CFU / mL), 1×10 7 CFU / g (CFU / mL), 5×10 7 CFU / g (CFU / mL), 1×10 8 CFU / g (CFU / mL), 3×10 8 CFU / g (CFU / mL), 5×10 8 CFU / g (CFU / mL), 1×10 9 CFU / g (CFU / mL), 3×10 9 CFU / g (CFU / mL), 5×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0014] Preferably, the dosage form of the preparation includes solution or powder.

[0015] The dosage form of the preparation for degrading nicotine involved in the present invention is not limited, including the most commonly used solution, freeze-dried powder, or further prepared capsules, tablets or granules.

[0016] In the present invention, the dosage form of the preparation is a solution, which is prepared by a method comprising the following steps:

[0017] The strain of Stenotrophomonas maltophilia YG-17 is inoculated into a culture medium and activated and fermented in sequence to obtain a fermentation liquid; the fermentation liquid is centrifuged, and the bacterial mud is resuspended with sterile water to obtain a bacterial suspension.

[0018] Preferably, the culture medium comprises LB medium.

[0019] Preferably, the fermentation culture is cultured at a temperature of 28-37° C. (e.g., 28° C., 29° C., 30° C., 32° C., 35° C., 37° C., etc.) for 10-30 hours (e.g., 10 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 30 hours, etc.). Other specific values ​​within the above numerical range can be selected and are not detailed here.

[0020] In a fourth aspect, the present invention provides a method for reducing nicotine contamination in tobacco waste, the method comprising: inoculating the Stenotrophomonas maltophilia YG-17 strain into the sterilized tobacco waste for fermentation.

[0021] Preferably, the inoculation rate of the Stenotrophomonas maltophilia YG-17 strain is not less than 1×10 8 CFU / g, for example 1×10 8 CFU / g, 1×10 9 CFU / g, 1×10 10 CFU / g, 5×10 10 CFU / g, 1×10 11 CFU / g, 5×10 11 CFU / g, 1×10 12 CFU / g, 3×10 12 CFU / g, 5×10 12 CFU / g, 1×10 13 CFU / g, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0022] Preferably, the tobacco waste is waste material generated during tobacco processing, production or use, including any one of tobacco waste, tobacco stems or tobacco rods, or a combination of at least two of them.

[0023] Preferably, the tobacco waste is crushed to 20-60 mesh before inoculation, such as 20 mesh, 25 mesh, 30 mesh, 35 mesh, 40 mesh, 45 mesh, 50 mesh, 55 mesh, 60 mesh, etc.

[0024] Preferably, the fermentation is carried out at a humidity of 40-55%, such as 40%, 42%, 44%, 45%, 48%, 50%, 53%, 55%, etc.

[0025] Preferably, the fermentation is first carried out at 27-33°C (e.g., 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, etc.) for 20-30h (e.g., 20h, 22h, 24h, 25h, 26h, 28h, 30h, etc.) and then at 35-40°C (e.g., 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.) for 20-30h (e.g., 20h, 22h, 24h, 25h, 26h, 28h, 30h, etc.).

[0026] All other specific point values ​​not listed in the numerical range of the present invention are within the protection scope of the present invention. Based on the consideration of brevity, they will not be described here one by one.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention isolated and preserved a new strain of Stenotrophomonas maltophilia that is capable of efficiently degrading nicotine from the soil of tobacco plant roots. Sequencing analysis revealed that the 16S rDNA sequence of the strain is shown in SEQ ID No: 1. A nucleic acid sequence alignment of the sequenced sequence revealed that the strain is Stenotrophomonas maltophilia. The strain has excellent nicotine-degrading ability, which makes it potentially useful in tobacco product production, reducing the health risks of tobacco products to humans. It can also be used to biodegrade nicotine-containing tobacco waste generated by the tobacco industry, promoting the recycling of waste resources. It can also be used to environmentally friendly treat nicotine contamination in soil or wastewater in tobacco-growing areas, contributing to environmental protection and avoiding environmental and ecological harm. Furthermore, the strain can enrich the resource of nicotine-degrading strains and be used to screen for genes related to efficient nicotine degradation, providing a new gene source for transgenic tobacco and engineered strains, thereby improving the quality of tobacco products and reducing the harm of nicotine to humans and the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the standard curve used in high performance liquid chromatography to detect nicotine content;

[0030] Figure 2 is a time course curve of nicotine degradation by Stenotrophomonas maltophilia YG-17;

[0031] The YG-17 strain involved in the present invention is classified as Stenotrophomonas maltophilia, with a deposit number of CCTCC NO: M 2025684 and a deposit date of April 2, 2025. The depository is China Center for Type Culture Collection, with an address of Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0033] Example 1

[0034] Isolation, screening and identification of Stenotrophomonas maltophilia YG-17:

[0035] (1) Preparation of culture medium:

[0036] LB medium: Dissolve 10 g of peptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of deionized water and autoclave at 121°C for 20 min. Solid LB medium is LB medium supplemented with 1.5% agar.

[0037] Nicotine medium: Dissolve 13.3g K2HPO4, 4g KH2PO4, 0.2g MgSO4·7H2O, and 0.5mL of trace element solution in 1L of deionized water. Autoclave at 121°C for 20 minutes, cool to 70°C, and add nicotine to the specified final concentration. For solid medium, add 1.5% agar to liquid medium. The trace element solution contains: 0.4g MnSO4·7H2O, 0.2g CaCl2·2H2O, and 0.2g FeSO4·7H2O. Bring to 100mL with distilled water.

[0038] (2) Enrichment of strains:

[0039] Soil samples were collected from the root zone of tobacco plants at a tobacco farm in Zunyi, Guizhou Province. Plant debris, tobacco shreds, and other debris were removed from the samples. 10.0 g of soil sample was accurately weighed and placed in a sterilized conical flask containing 100 mL of sterile water. The sample was then placed in a shaking incubator at 30°C for 8 hours, removed, and allowed to stand for 30 minutes to obtain a soil suspension. 1 mL of the supernatant of the soil suspension was added to 100 mL of a culture medium containing 1 g / L nicotine. The suspension was then incubated at 30°C with shaking for 3 days. After the incubation period, the sample was removed and allowed to stand for 30 minutes to obtain an enrichment suspension.

[0040] (3) Screening of strains:

[0041] Take an appropriate amount of enriched suspension and dilute it by 10-3 , 10 -4 and 10 -5 100 μL of bacterial suspension at different concentrations was spread onto a solid nicotine medium with a nicotine content of 1 g / L and incubated at 30°C in the dark for 5 days. Single colonies of different morphologies on the nicotine medium were selected and streaked onto a solid nicotine medium with a nicotine content of 1 g / L for further isolation and purification. The purified single colonies were then spread and cultured on solid nicotine medium with increasing nicotine concentrations, such as 2 g / L and 3 g / L. After screening, a strain with the best nicotine tolerance was obtained and named YG-17.

[0042] (4) Identification of strains:

[0043] The YG-17 single bacterium was picked and inoculated into LB medium and cultured for 12 hours to obtain a bacterial solution. DNA extraction was performed using a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). The extracted genomic DNA was amplified by PCR using universal PCR primers. After the PCR product was detected by 1% agarose gel electrophoresis, Shanghai Shenggong Gene was commissioned to perform bidirectional sequencing of the sequence. The 16S rDNA gene sequence of the strain is shown in SEQ ID No: 1, and it was submitted to NCBI for BLAST homology similarity comparison to determine its species. The strain was identified as Stenotrophomonas maltophilia.

[0044] SEQ ID No: 1:

[0045]

[0046] Example 2

[0047] Evaluation of the nicotine degradation effect of YG-17 strain:

[0048] (1) Preparation of bacterial suspension: The activated Stenotrophomonas maltophilia YG-17 was inoculated into LB liquid culture medium (prepared in the same manner as in Example 1), and cultured in a shaking incubator at 30°C and 200 rpm for 18 h. The bacterial suspension was cultured until the OD 600 The concentration of bacterial suspension was 6 × 10 7 CFU / mL.

[0049] (2) 100 μL of the YG-17 bacterial suspension was inoculated into 10 mL of a 2 g / L liquid nicotine culture medium (prepared as in Example 1) at 30°C and shaken at 200 r / min. At 6, 12, 18, 24, and 30 h of incubation, 100 μL of the fermentation broth was taken, diluted 10-fold with 40% methanol, centrifuged, and 1 mL of the supernatant was filtered through a 0.22 μm filter membrane. The nicotine content was determined by high-performance liquid chromatography. 10 mL of a blank liquid culture medium without nicotine was used as a control to calculate the nicotine content and determine the degradation rate of the degrading strain.

[0050] The nicotine content is detected by high performance liquid chromatography, and the nicotine content in the culture medium or culture to be tested is calculated based on the nicotine standard curve. The high performance liquid chromatography detection conditions are: SinoChrom ODS-BP C18 column (250mm×4.6mm, 5μm); column temperature 40°C; ambient temperature 20-26°C; mobile phase is a mixture of methanol and 0.02mol / L phosphate buffer (pH 6.5) containing 0.4% triethylamine in a volume ratio of 40:60; flow rate 1.0ml / min; injection volume 10μL; detection wavelength 259nm. The external standard method is quantified by peak area. The nicotine standard curve is as follows: Figure 1 As shown, the obtained linear regression equation is y=9969.4x+15828, the correlation coefficient is 0.9999, and the linear range is 1.0-1000.0 μg / mL, where y is the peak area and x is the nicotine content.

[0051] The nicotine content test results and nicotine degradation rate results at different times are as follows Figure 2 and as shown in Table 1.

[0052] Table 1

[0053] time Degradation rate 6h 9.0％ 12h 54.5％ 18h 100.0％ 24h 100.0％ 30h 100.0％

[0054] Depend on Figure 2As shown in Table 1, when the YG-17 strain was used to treat a 2 g / L liquid nicotine culture medium and cultured on a shaking table for 12 h, the nicotine degradation rate could reach 54.5%. After 18 h of culture on a shaking table, the nicotine could be completely degraded (degradation rate ≥ 99%). Its degradation efficiency was significantly higher than that of similar strains reported previously. Therefore, the strain YG-17 has a very excellent ability to degrade nicotine.

[0055] Example 3

[0056] Evaluation of the effect of YG-17 strain on degrading nicotine in tobacco waste:

[0057] (1) Preparation of cigarette waste samples:

[0058] Dried discarded tobacco dust, tobacco rods, and tobacco stems (all provided by Nanjing Cigarette Factory of Jiangsu China Tobacco Industrial Co., Ltd.) were mixed in a ratio of 1:1:1 by mass, crushed with a pulverizer, and passed through a 40-mesh sieve for later use.

[0059] (2) Fermentation treatment of tobacco waste:

[0060] Weigh 2g of tobacco waste sample and place it in a 100mL conical flask, sterilize it at 121℃ for 20min, and then cool it to 25℃. YG-17 bacterial suspension (prepared as in Example 2) was prepared at 4×10 8 The CFU / g inoculum was inoculated into the sterilized conical flask of tobacco waste, and then 1 mL of sterile water was added to rinse the bacteria and the wall of the centrifuge tube and added to the conical flask, so that the humidity of the tobacco waste was maintained at 40-55%. A sterile glass rod was used to mix the bacteria and solid tobacco waste evenly. The conical flask was first placed at 30°C for 24 hours and then at 37°C for 24 hours.

[0061] After fermentation, 0.3% NaOH solution was added at a material-liquid ratio of 1:22.5, and nicotine was extracted by ultrasonication at 40°C for 40 minutes. After ultrasonication, the mixture was centrifuged at 10,000 rpm for 2 minutes. The supernatant was diluted with 40% methanol, filtered through a 0.22 μm organic filter, and tested by HPLC under the same conditions as in Example 2.

[0062] The results showed that the initial nicotine content of the tobacco waste sample was 1.78%. After 48 hours of fermentation with the YG-17 strain, the nicotine degradation rate reached more than 99% (undetectable).

[0063] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0064] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0065] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A strain of Stenotrophomonas maltophilia YG-17 that efficiently degrades nicotine, characterized in that: The classification name of the Stenotrophomonas maltophilia YG-17 is Stenotrophomonas maltophilia, the preservation number is CCTCC NO: M 2025684, and the preservation date is April 2, 2025.

2. Use of the Stenotrophomonas maltophilia YG-17 or its fermentation product according to claim 1 in the preparation of a nicotine degrader.

3. A preparation for degrading nicotine, characterized in that: The strain in the preparation for degrading nicotine includes the Stenotrophomonas maltophilia YG-17 according to claim 1.

4. The preparation for degrading nicotine according to claim 3, characterized in that The number of viable bacteria of Stenotrophomonas maltophilia YG-17 in the preparation is not less than 6×10 6 CFU / mL or 6×10 6 CFU / g.

5. The preparation for degrading nicotine according to claim 3, characterized in that: The dosage form of the preparation includes solution or powder.

6. The preparation for degrading nicotine according to claim 3, characterized in that: The dosage form of the preparation is a solution, which is prepared by a method comprising the following steps: The strain of Stenotrophomonas maltophilia YG-17 is inoculated into a culture medium and activated and fermented in sequence to obtain a fermentation liquid; the fermentation liquid is centrifuged, and the bacterial mud is resuspended with sterile water to obtain a bacterial suspension.

7. A method for reducing nicotine pollution in tobacco waste, characterized in that: The method comprises: inoculating the Stenotrophomonas maltophilia YG-17 strain into sterilized tobacco waste for fermentation.

8. The method according to claim 7, characterized in that The inoculation rate of the Stenotrophomonas maltophilia YG-17 strain is not less than 1×10 8 CFU / g; Preferably, the tobacco waste is waste material generated during tobacco processing, production or use, including any one of tobacco waste, tobacco stems or tobacco rods, or a combination of at least two thereof; Preferably, the tobacco waste is crushed to 20-60 mesh before inoculation.

9. The method according to claim 7, characterized in that The fermentation is carried out at a humidity of 40-55%.

10. The method according to claim 7, characterized in that The fermentation is first carried out at 27-33°C for 20-30 hours, and then at 35-40°C for 20-30 hours.

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