Ensifer mucilaginosus YG1-4-1 with nicotine degradation effect and application thereof

Abstract

The invention relates to an ensifer mucilaginosus YG1-4-1 with a nicotine degrading effect and application of the ensifer mucilaginosus YG1-4-1. The preservation number of the ensifer mucilaginosus YG1-4-1 is CCTCC (China Center for Type Culture Collection) NO: M 2025683. The strain has excellent nicotine degradation capability, has the capability of growing by taking nicotine as a unique carbon and nitrogen source, and can completely degrade nicotine after being cultured in a culture medium containing 2g / L nicotine for 30 hours; the strain is inoculated in tobacco waste and cultured for 2 days, and the degradation rate of nicotine in tobacco waste can reach 99% or above (the nicotine cannot be detected by high performance liquid chromatography). The strain enriches resources of nicotine degrading strains, the degrading process of the strain has high efficiency and safety, and the strain has wide application potential in the fields of green production of tobacco industry and environmental pollution control.

Description

Technical Field

[0001] The present invention belongs to the technical field of strain development, and relates to a new strain capable of degrading nicotine and its application, in particular to a nicotine-degrading Xanthophyll sp. YG1-4-1 strain and its application. Background Art

[0002] Tobacco (Nicotiana tabacum L.) is a major cash crop in my country. Its plants contain alkaloids such as nicotine and nornicotine, with nicotine accounting for over 94% of the total. Nicotine, a weakly basic compound composed of pyridine and pyrrole dinitrogen heterocycles, is a core component of tobacco addiction and is toxic to humans. Nicotine can also damage ecosystems and cause environmental pollution through smoke and wastewater. Nicotine pollution is caused by tobacco waste residues, combustion release, and the use of neonicotinoid pesticides.

[0003] Nicotine degradation technologies are primarily categorized into agricultural techniques, physical methods, chemical oxidation, and microbial degradation. Agricultural techniques achieve nicotine control through conventional breeding to select low-nicotine varieties, gene editing to manipulate metabolic pathways, and cultivation management (such as optimizing fertilization and harvesting times). However, these techniques present challenges such as complex gene regulation, high environmental sensitivity, and difficulty balancing economic benefits with industrial needs. Among physical methods, solvent extraction is limited by high energy consumption and process complexity, while chemical oxidation methods (such as photocatalysis and Fenton oxidation) face challenges such as high energy consumption and byproduct contamination.

[0004] Microbial degradation has become a research hotspot in recent years due to its highly specific and environmentally friendly properties. Since Wada et al. isolated a nicotine-degrading bacterium (Pseudomonas sp. 41) from tobacco leaves in 1953, a growing number of microbial strains with nicotine-degrading capabilities have been discovered, most of which are bacteria, such as Ochrobactrum, Arthrobacter, and Pseudomonas. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a new strain with the ability to degrade nicotine and its application, in particular to provide a sticky sword fungus YG1-4-1 with the ability to degrade nicotine and its application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an Ensifer adhoerens YG1-4-1 having the efficacy of degrading nicotine. The classification name of the Ensifer adhoerens YG1-4-1 is Ensifer adhoerens (recorded as Ensifer adhoerens in the strain preservation certificate, but actually should be Ensifer adhaerens), the preservation number is CCTCC NO: M 2025683, and the preservation date is April 2, 2025.

[0008] The present invention has isolated and preserved a new strain of Xanthoceras glutinosus that can efficiently degrade nicotine from the root soil of tobacco plants. The strain was sequenced and analyzed, and its 16S rDNA sequence was shown as SEQ ID No: 1. The sequence obtained by sequencing was compared with the nucleic acid sequence, and the result showed that the strain was Xanthoceras glutinosus. The strain has excellent nicotine degradation ability and has the ability to grow with nicotine as the only carbon and nitrogen source. It can completely degrade nicotine after being cultured in a culture medium containing 2g / L nicotine for 30h. The strain was inoculated into tobacco waste and cultured for 2 days, and the nicotine degradation rate in the tobacco waste can reach more than 99% (undetectable by high-performance liquid chromatography). The strain enriches the resources of nicotine-degrading strains, and its degradation process is both efficient and safe, showing broad application potential in the fields of green production and environmental pollution control in the tobacco industry.

[0009] In a third aspect, the present invention provides use of the Xanthoceras glutinosus YG1-4-1 or a fermentation product thereof described in the first aspect in preparing a product for degrading nicotine.

[0010] In a third aspect, the present invention provides a bacterial agent for degrading nicotine, wherein the strain in the bacterial agent for degrading nicotine includes the Xanthophyllales strain YG1-4-1 described in the first aspect.

[0011] Preferably, the bacterial agent is in the form of a solution or a powder.

[0012] 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.

[0013] Preferably, the number of viable bacteria of the sticky sword fungus YG1-4-1 in the bacterial agent is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g, for example 1×10 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 8CFU / 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] In the present invention, the formulation of the bacterial agent is a solution, which is prepared by a method comprising the following steps:

[0015] The YG1-4-1 strain of the sticky sword fungus 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.

[0016] In a fourth aspect, the present invention provides a method for degrading nicotine in tobacco waste, the method comprising: inoculating the Xanthophyllales strain YG1-4-1 into the sterilized tobacco waste for culturing.

[0017] Preferably, the inoculation amount of the YG1-4-1 strain of the sticky sword fungus 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.

[0018] 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.

[0019] 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.

[0020] Preferably, the culturing is carried out at a humidity of 30-50%, for example, 30%, 32%, 35%, 38%, 40%, 42%, 45%, 50%, etc.

[0021] Preferably, the culture 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 18-36h (e.g., 18h, 20h, 22h, 24h, 25h, 26h, 28h, 30h, 36h, etc.), and then at 35-40°C (e.g., 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.) for 18-36h (e.g., 18h, 20h, 22h, 24h, 25h, 26h, 28h, 30h, 36h, etc.).

[0022] 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.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention has isolated and preserved a new strain of Xanthoceras glutinosus that can efficiently degrade nicotine from the root soil of tobacco plants. The strain was sequenced and analyzed, and its 16S rDNA sequence was shown as SEQ ID No: 1. The sequence obtained by sequencing was compared with the nucleic acid sequence, and the result showed that the strain was Xanthoceras glutinosus. The strain has excellent nicotine degradation ability and has the ability to grow with nicotine as the only carbon and nitrogen source. It can completely degrade nicotine after being cultured in a culture medium containing 2g / L nicotine for 30h. The strain was inoculated into tobacco waste and cultured for 2 days, and the nicotine degradation rate in the tobacco waste can reach more than 99% (undetectable by high-performance liquid chromatography). The strain enriches the resources of nicotine-degrading strains, and its degradation process is both efficient and safe, showing broad application potential in the fields of green production and environmental pollution control in the tobacco industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the bacterial genus relationship diagram detected after 16s rDNA sequencing of YG1-4-1 bacteria;

[0026] Figure 2 is a time course curve of nicotine degradation by strain YG1-4-1;

[0027] The YG1-4-1 strain involved in the present invention is classified and named Ensifer adhoerens, with a deposit number of CCTCC NO: M 2025683 and a deposit date of April 2, 2025. The depository is China Center for Type Culture Collection, and the address is: Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1

[0030] Isolation, screening and identification of Xanthophyllum adhensum YG1-4-1:

[0031] (1) Preparation of culture medium:

[0032] 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.

[0033] 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. Solid medium consists of liquid medium supplemented with 1.5% agar. For the trace element solution, dissolve 0.4g MnSO4·7H2O, 0.2g CaCl2·2H2O, and 0.2g FeSO4·7H2O in 0.1mol / L HCl and dilute to 100mL.

[0034] (2) Enrichment of strains:

[0035] 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 6 hours and allowed to stand for 30 minutes to obtain a soil suspension. 1 mL of the supernatant from 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.

[0036] (3) Screening of strains:

[0037] Take an appropriate amount of enriched suspension and dilute it by 10 -3 , 10 -4 and 10 -5100 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 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 content. After screening, a strain with good nicotine tolerance was obtained and named YG1-4-1.

[0038] (4) Identification of strains:

[0039] Pick up the YG1-4-1 single bacterium and inoculate it into LB medium for 12 hours to obtain a bacterial solution. Use a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract DNA. 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. It was submitted to NCBI for BLAST homology similarity comparison to determine its species. The strain was identified as Adhesive Sword Bacteria. Download the strain sequences with higher BLAST sequence homology, use MEGA5.05 software to calculate the sequence similarity, and use the Neighbor-Joining method to construct the strain phylogenetic tree, as shown below: Figure 1 As shown, the phylogenetic position of the species was determined.

[0040] SEQ ID No: 1:

[0041]

[0042] Example 2

[0043] Evaluation of the nicotine degradation effect of strain YG1-4-1:

[0044] (1) Preparation of bacterial suspension: The activated sticky sword YG1-4-1 was inoculated into LB liquid culture medium (prepared in the same way as in Example 1), and cultured on a shaking platform at 30°C and 180 rpm for 18 h. The bacterial suspension was cultured until the OD 600 The concentration of bacterial suspension was 6 × 10 6 CFU / mL.

[0045] (2) 100 μL of the YG1-4-1 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 and 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.

[0046] Nicotine content was determined using high-performance liquid chromatography (HPLC). The nicotine content in the culture medium or culture to be tested was calculated based on a nicotine standard curve. HPLC conditions were: SinoChrom ODS-BP C18 column (250 mm × 4.6 mm, 5 μm); column temperature 40°C; ambient temperature 20-26°C; mobile phase consisting of a 40:60 (volume ratio) mixture of methanol and 0.02 mol / L phosphate buffer (pH 6.5) containing 0.4% triethylamine; flow rate 1.0 ml / min; injection volume 10 μL; detection wavelength 259 nm. Quantification was performed using the external standard method based on peak area.

[0047] The nicotine content test results and nicotine degradation rate results at different times are as follows Figure 2 and as shown in Table 1.

[0048] Table 1

[0049] time Degradation rate 6h 0.0％ 12h 13.5％ 18h 39.5％ 24h 81.5％ 30h 100.0％

[0050] Depend on Figure 2As shown in Table 1, when the YG1-4-1 strain was used to treat a 2 g / L liquid nicotine culture medium, the nicotine degradation effect increased with the increase of treatment time. When the culture time reached 30 h, nicotine could be completely degraded. Therefore, the YG1-4-1 strain had a very excellent ability to degrade nicotine.

[0051] Example 3

[0052] Evaluation of the effect of strain YG1-4-1 on degrading nicotine in tobacco waste:

[0053] (1) Preparation of cigarette waste samples:

[0054] 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.

[0055] (2) Fermentation treatment of tobacco waste:

[0056] 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℃. YG1-4-1 bacterial suspension (prepared as in Example 2) was prepared at 4×10 8 The CFU / g inoculum was inoculated into a 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 to maintain the humidity of the tobacco waste at 30-50%. 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.

[0057] 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.

[0058] The results showed that the initial nicotine content of the tobacco waste sample was 1.78%. After 48 hours of fermentation with the YG1-4-1 strain, the nicotine degradation rate reached more than 99% (undetectable).

[0059] 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.

[0060] 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.

[0061] 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 nicotine-degrading fungus YG1-4-1, characterized in that: The classification name of the adhesin sword fungus YG1-4-1 is Ensifer adhoerens, the preservation number is CCTCC NO: M2025683, and the preservation date is April 2, 2025.

2. Use of the Xanthoceras glutinosus YG1-4-1 or its fermentation product according to claim 1 in the preparation of products for degrading nicotine.

3. A bacterial agent for degrading nicotine, characterized in that: The strain in the bacterial agent for degrading nicotine includes the sticky sword fungus YG1-4-1 described in claim 1.

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

5. The bacterial agent for degrading nicotine according to claim 3, characterized in that: The number of viable bacteria of the sticky sword fungus YG1-4-1 in the bacterial agent is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

6. The bacterial agent for degrading nicotine according to claim 3, characterized in that: The formulation of the bacterial agent is a solution, which is prepared by a method comprising the following steps: The YG1-4-1 strain of the sticky sword fungus 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 degrading nicotine in cigarette waste, characterized in that: The method comprises the following steps: inoculating the YG1-4-1 strain of Xanthophyllales ctenosarum into sterilized tobacco waste for culturing.

8. The method according to claim 7, characterized in that The inoculation amount of the YG1-4-1 strain of the sticky sword fungus 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 culture was carried out at a humidity of 30-50%.

10. The method according to claim 7, characterized in that The culture is first carried out at 27-33°C for 18-36 hours, and then at 35-40°C for 18-36 hours.

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