Pseudomonas strain TR14 and its use in degrading nicotine
By screening and identifying the Pseudomonas strain TR14, the problem of weak degradation ability of existing microbial degrading strains has been solved, achieving the effect of efficient degradation of nicotine and improvement of tobacco leaf quality, which is applicable to the tobacco industry and environmental protection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHINA TOBACCO ANHUI IND CO LTD
- Filing Date
- 2024-12-17
- Publication Date
- 2026-05-22
AI Technical Summary
Existing microbial degrading strains have a weak ability to degrade nicotine, a low tolerance for nicotine concentrations, and some bacteria only degrade nicotine, making it difficult to guarantee various quality indicators of tobacco and thus unable to be widely used in actual production.
Pseudomonas strain TR14 was screened from tobacco soil in Anhui Province. The strain was obtained through enrichment culture and purification, and molecular identification was performed. It was then prepared into a microbial agent for nicotine degradation. It is suitable for different grades of tobacco leaves and has a good nicotine degradation effect. At the same time, it can reduce the protein and starch content in tobacco leaves and inhibit mold growth.
The Pseudomonas strain TR14 exhibits 100% degradation of nicotine at 32℃ and pH 7.0, significantly improving tobacco quality, reducing nicotine, protein, and starch content, decreasing irritation, increasing aroma, and demonstrating good antibacterial effects. It is suitable for the tobacco industry and environmental protection.
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Abstract
Description
Technical Field
[0001] This invention relates to a Pseudomonas strain TR14 and its application in the degradation of nicotine, belonging to the field of microbial application technology. Background Technology
[0002] Nicotine, also known as tobacco alkaloid, is the main alkaloid found in tobacco, accounting for over 95% of the total alkaloid content. It is also one of the main harmful components in cigarettes and tobacco leaves. Nicotine is highly toxic and addictive; a single intake of 4-8 mg can cause poisoning in adults, while a single intake of 40-60 mg can be fatal in children. Large doses of nicotine can suppress the central nervous system, causing cardiac arrest, and in severe cases, can be fatal. During smoking, nicotine is also nitrified, producing nitrosamines (TSNAs), a potent carcinogen unique to tobacco, which causes significant harm to the human body. Nicotine not only has toxic and carcinogenic side effects on humans, but it also affects soil ecological structure, pollutes groundwater, and causes serious soil and environmental pollution, disrupting the ecological balance. Therefore, reducing the nicotine content in tobacco leaves and the environment is of paramount importance for maintaining human health and protecting the ecological environment.
[0003] Currently, nicotine degradation mainly focuses on biological treatment. Compared with physical and chemical treatment methods, microbial degradation of nicotine has advantages such as strong degradation ability, rich variety of functional microorganisms, diverse degradation pathways, and minimal environmental impact. Microbial degradation of nicotine is considered a relatively economical and effective method. Numerous studies have shown that utilizing microorganisms to degrade nicotine has significant implications and promising prospects in reducing tobacco harm, protecting the environment, and improving the utilization rate of tobacco resources. Chinese patent application No. 202310934714.9 discloses a strain of *Pseudomonas hunanensis* MGJ-2 and its application in nicotine degradation, with accession number CGMCCNo.25121. After treating nicotine-inorganic salt culture medium with strain MGJ-2 for 25 hours, the degradation rate of 500 mg / L nicotine was 99.9%. Chinese patent application No. 200710070805.3 discloses a new nicotine-degrading bacterium—*Pseudomonas hunanensis* ZUTSKD—and its applications. This bacterium can degrade nicotine, with a maximum tolerable nicotine concentration of 5.5 g / L. Chinese patent CN113462611A discloses a *Arthrobacter urealyticum* strain for degrading nicotine and its applications. However, the efficiency of nicotine degradation is low; after 104 hours of cultivation, 2 g / L of nicotine can be completely degraded.
[0004] The aforementioned strains exhibit effective degradation capabilities for nicotine in tobacco leaves. However, the nicotine degradation ability of microbial degrading bacteria is not very strong, and their tolerance to nicotine concentrations is generally low. Furthermore, some existing bacteria only degrade nicotine and cannot guarantee various quality indicators of tobacco, making them difficult to apply in actual production. Therefore, there is an urgent need to cultivate strains that can more effectively degrade nicotine, coordinate tobacco quality, and have broader application value. Summary of the Invention
[0005] Currently, microbial nicotine-degrading bacteria have weak nicotine degradation capabilities and low tolerance to nicotine concentrations. Furthermore, some bacteria only degrade nicotine, failing to guarantee various quality indicators of tobacco. To address these issues, this invention screened highly efficient nicotine-degrading strains from tobacco soil in Anhui Province and prepared them into a microbial agent. This agent exhibits good versatility in reducing nicotine in tobacco leaves, showing significant nicotine reduction effects on upper tobacco leaves at different grades. Simultaneously, the strain demonstrates strong overall performance, reducing the protein and starch content of tobacco leaves, and the fermentation broth also exhibits good inhibitory effects on tobacco molds.
[0006] The present invention relates to a Pseudomonas strain TR14, which has been deposited at the China Center for Type Culture Collection (CCTCC) under the classification name Pseudomonas sp. TR14, deposited on November 8, 2024, with accession number CCTCC NO: M 20242492. The strain was obtained from soil from tobacco fields in Jingxian County, Xuancheng City, Anhui Province, where tobacco is cultivated year-round.
[0007] The method for isolating and purifying the pseudomonad strain TR14 of the present invention includes the following steps:
[0008] Step 1: Take 5 g of tobacco field soil and add it to an Erlenmeyer flask containing 100 mL of enrichment medium. Incubate at 28℃ and 200 rpm for 48 h on a shaker. Take 1 mL of bacterial suspension and transfer it to another Erlenmeyer flask containing 100 mL of enrichment medium. Continue to incubate under the same conditions. Repeat the incubation 3 times to achieve the purpose of enrichment culture.
[0009] Step 2: Dilute the fermentation broth by 10 1 -10 4 The culture medium was spread onto the selected medium and cultured at 28°C for 48 h. Nicotine-tolerant strains were obtained by screening and then inoculated onto LB solid medium for purification. The obtained single colonies were stored in glycerol tubes at 4°C.
[0010] The enrichment medium is formulated as follows: K2HPO4 13.3 g / L, KH2PO4 4 g / L, (NH4)2SO4 0.1 g / L, yeast extract 1.0 g / L, trace element solution 10 mL / L, pH 7.0, autoclaved at 121℃ for 20 min, and nicotine was added after being sterilized by filtration through a 0.22 µm filter membrane, with a nicotine content of 0.5 g / L.
[0011] The trace element solution is formulated as follows: Weigh 1.0 g of MgSO4·7H2O, 0.4 g of MnSO4·H2O, 0.2 g of CaCl2·2H2, 0.2 g of CuCl2·2H2O, and 0.02 g of FeSO4·7H2O, dissolve them in 0.1 mol / L HCl solution, and bring the volume to 100 mL.
[0012] The screening medium was prepared based on the enrichment medium formula, with an additional 1.5% agar powder added and the nicotine content adjusted to 1.0 g / L.
[0013] LB medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, sodium chloride 5.0 g / L, pH 7.0. Autoclave at 121℃ for 20 min. Add 1.5% agar to the solid medium.
[0014] The molecular identification steps of the Pseudomonas strain of this invention are as follows:
[0015] The purified strain was inoculated onto LB agar and incubated at 28°C for 24 hours. Molecular identification of the purified strain was performed: the 16S rRNA gene sequence of strain TR14 was amplified by PCR using universal bacterial primers. The amplification product was subjected to 1% agarose gel electrophoresis, and the target band (approximately 1500 bp) was recovered according to the Easy Pure Quick Gel Extraction Kit instructions. Simultaneously, the 16S rRNA sequence of strain TR14 was aligned using the EzBioCloud website, and *Pseudomonas* species with high sequence correlation were selected for phylogenetic analysis. A phylogenetic tree was constructed using the Mega 4.0 program.
[0016]
[0017] The application of the pseudomonad strain TR14 in the degradation of nicotine is described in this invention.
[0018] Pseudomonas strain TR14 was inoculated at a 1% inoculation rate into enrichment medium containing nicotine (0.5 g / L) and cultured for 24 h. TR14 cells continued to proliferate within 3-12 h, with a slight decrease in cell concentration between 12-24 h. The maximum OD value was observed during the 12 h culture period. 600 =1.05767. Within 12 hours, strain TR14 achieved 100% maximum degradation capacity for nicotine. This indicates that the TR14 strain isolated and identified in this invention has a nicotine-degrading effect.
[0019] Furthermore, the temperature of the degradation system is 23℃~37℃, with the optimal temperature being 32℃; the pH of the degradation system is 6.0~9.0, preferably 7.0.
[0020] Preparations of nicotine-degrading formulations were prepared using the aforementioned Pseudomonas strain TR14 or its bacterial culture.
[0021] Furthermore, a concentration of 1×10 was used. 7 ~1×10 9 Tobacco was sprayed with a bacterial solution of Pseudomonas strain TR14 at CFU / mL.
[0022] Furthermore, the spraying rate is 15-25% (w / w), and after spraying, the plants are cultured at 28℃ and 65% humidity for 14-16 days under controlled temperature and humidity conditions.
[0023] The application of the Pseudomonas strain TR14 in the preparation of antibacterial agents.
[0024] The antibacterial agent has a good inhibitory effect on tobacco mold.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] This invention provides a nicotine-degrading Pseudomonas TR14 strain with high biological activity, simple cultivation, and stable genetic properties. Research shows that Pseudomonas TR14 exhibits good nicotine degradation at 32℃ and pH 7.0. Using this invention to improve the quality of tobacco raw materials, especially upper tobacco leaves, involves a simple process and mild reaction conditions. After treatment, the nicotine, protein, and starch content of tobacco leaves is reduced, irritation is significantly reduced, the smoke is smoother, the aroma is increased, and the industrial usability of tobacco leaves is significantly improved. Pseudomonas TR14 also has a good inhibitory effect on tobacco molds. The TR14 strain can be used as a novel microbial agent for degrading the nicotine content of tobacco, showing great application potential. Attached Figure Description
[0027] Figure 1 This is a phylogenetic tree diagram of TR14.
[0028] Figure 2 This is the result of the TR14 genus comparison and identification in this invention.
[0029] Figure 3 The degradation ability of strain TR14 for nicotine.
[0030] Figure 4 The graph shows the effect of nicotine concentration on the degradation of nicotine by strain TR14.
[0031] Figure 5 This is a diagram showing the results of strain TR14 antagonizing Tobacco Leaf Mold. Detailed Implementation
[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments.
[0033] The present invention relates to a Pseudomonas strain TR14, classified as Pseudomonas sp. TR14, which was deposited at the China Center for Type Culture Collection (CCTCC) on November 8, 2024, with accession number CCTCC NO: M 20242492. The strain was obtained from soil from tobacco fields in Jingxian County, Xuancheng City, Anhui Province, where tobacco is cultivated year-round.
[0034] Example 1: Isolation and identification of nicotine-degrading microorganisms
[0035] The method for isolating Pseudomonas strains for nicotine degradation includes the following steps:
[0036] Preparation of culture medium:
[0037] Enrichment medium composition: K₂HPO₄ 13.3 g / L, KH₂PO₄ 4 g / L, (NH₄)₂SO₄ 0.1 g / L, yeast extract 1.0 g / L, trace element solution 10 mL / L, pH 7.0. After autoclaving at 121℃ for 20 min, nicotine was added as needed after sterilization by filtration through a 0.22 µm filter membrane.
[0038] Trace element solution: Weigh 1.0 g of MgSO4·7H2O, 0.4 g of MnSO4·H2O, 0.2 g of CaCl2·2H2, 0.2 g of CuCl2·2H2O, and 0.02 g of FeSO4·7H2O, dissolve and dilute to 100 mL with 0.1 mol / L HCl solution.
[0039] Isolation medium: Add 1.5% agar powder to the enrichment medium.
[0040] LB medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, sodium chloride 5.0 g / L, pH 7.0. Autoclave at 121℃ for 20 min. Add 1.5% agar to the solid medium.
[0041] Isolation and purification of nicotine-degrading bacteria: 5 g of tobacco soil was added to an Erlenmeyer flask containing 100 mL of enrichment medium (nicotine content 0.5 g / L) and cultured at 28℃ and 200 rpm for 48 h on a shaker. 1 mL of the bacterial suspension was transferred to another Erlenmeyer flask containing 100 mL of enrichment medium, and cultured under the same conditions for three consecutive times to achieve enrichment. The fermentation broth was then diluted and spread onto screening medium (nicotine content 1.0 g / L), and cultured at 28℃ for 48 h. After enrichment and isolation, bacteria tolerant to nicotine were screened and designated TR14. These were then inoculated onto LB solid medium for purification, and the obtained single colonies were preserved in glycerol tubes and stored at 4℃.
[0042] Molecular identification steps for Pseudomonas strains:
[0043] The purified strain was inoculated onto LB solid medium and cultured at 28°C for 24 h. Molecular identification of the purified strain was performed: the 16S rRNA gene sequence of strain TR14 was amplified by PCR using universal bacterial primers. The amplified product was subjected to 1% agarose gel electrophoresis, and the target band (approximately 1500 bp) was recovered according to the Easy Pure Quick Gel Extraction Kit instructions. Simultaneously, the 16S rRNA sequence of strain TR14 was aligned using the EzBioCloud website, and *Pseudomonas* species with high sequence relevance were selected for phylogenetic analysis. A phylogenetic tree was constructed using the Mega4.0 program. The amplified sequence was sequenced, and the similarity of the 16S rRNA gene sequences of the strains was compared in a database. Strains with a similarity higher than 98.65% were considered to be of the same genus. The results showed that strain TR14 belongs to the genus *Pseudomonas* (…). Figure 2 ), such as phylogenetic tree Figure 1 As shown.
[0044] The 16S rRNA nucleotide sequence of the strain is shown below:
[0045]
[0046] The aforementioned Pseudomonas strain is used to degrade nicotine.
[0047] Example 2: Relationship between nicotine degradation rate and growth rate of strain TR14
[0048] The TR14 glycerol strain, preserved at -80℃, was inoculated into fresh liquid LB medium at a 1:100 ratio and activated at 37℃ for 12 h at 200 rpm to obtain seed culture. The seed culture was then inoculated into fresh liquid LB medium at a 1:100 ratio and cultured with shaking at 37℃ for 200 rpm until the logarithmic growth phase. The bacterial cells were collected by centrifugation at 12000 rpm for 5 min, resuspended in an equal volume of enrichment medium, centrifuged again, washed twice, and resuspended in enrichment medium to obtain the degrading seed culture TR14.
[0049] The activated and degraded strain TR14 was inoculated at a 1% inoculation rate into a nicotine-enriched medium (0.5 g / L) and cultured for 24 h at pH 7.0, 32℃, and 200 rpm, with an uninoculated nicotine-containing medium as a control. Samples were taken at 3 h, 6 h, 9 h, 12 h, 15 h, 18 h, and 24 h, and the absorbance of strain TR14 at OD 600 nm was measured spectrophotometrically. Simultaneously, culture media at 3 h, 6 h, 9 h, 12 h, 15 h, 18 h, and 24 h were centrifuged at 12000 rpm for 10 min at 4℃, and the supernatant was collected and diluted with 0.05 mol / L HCl to an appropriate absorbance range. Using 0.05 mol / L HCl as a reference solution, the changes in the characteristic absorption peak of nicotine at 260 nm were measured.
[0050] The results are as follows Figure 3 As shown, strain TR14 cells continued to proliferate within 3-12 h, and the cell concentration decreased within 12-24 h. The maximum OD was observed during the 12 h culture period. 600 =1.05767. The nicotine concentration in the culture medium decreased significantly after 6 h, and the maximum degradation capacity of strain TR14 for nicotine reached 100% within 12 h. This indicates that the TR14 strain isolated and identified in this invention has a nicotine-degrading effect.
[0051] Example 3: The rate of nicotine degradation by strain TR14 under different culture conditions (temperature, pH, nicotine concentration, etc.)
[0052] TR14 strain cell suspension (OD) 600 1) Inoculate into 30 mL of basal culture medium and culture. Take samples regularly to determine the bacterial growth and nicotine degradation. Repeat each experiment 3 times.
[0053] Culture temperature: Nicotine concentration was 0.5 g / L, and the culture temperature was 23, 28, 32, 37 and 42℃, pH 7.0 and 200 rpm.
[0054] Initial pH of the culture medium: The initial pH values were 5, 6, 7, 8 and 9, and the nicotine concentration was 0.5 g / L. The medium was cultured at 32℃ and 200 rpm.
[0055] Initial nicotine concentrations: Nicotine concentrations of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 g / L were used for culturing at pH 7.0, 32℃, and 200 rpm.
[0056] The rate of nicotine degradation by strain TR14 was determined under different temperature, pH, and isostatic cultivation conditions for 12 h. The results are shown in Table 2. Strain TR14 can grow in the range of 23 ℃ to 37 ℃, and grows well within this range. The optimal growth temperature for strain TR14 is 32 ℃, at which temperature it reaches its maximum growth and highest nicotine degradation rate. The results are shown in Table 3. Strain TR14 grows well in the pH range of 6.0 to 9.0. The optimal pH for cell growth is 7.0, under which condition strain TR14 reaches its maximum growth and also exhibits the highest nicotine degradation rate.
[0057]
[0058] The rate of nicotine degradation by strain TR14 under different nicotine concentrations and the same culture conditions for 24 h was determined. Figure 4 The results showed that when the nicotine concentration exceeded 7.0 g / L, the TR14 strain degraded nicotine slowly. At a nicotine concentration of 7.0 g / L, the TR14 strain achieved a nicotine degradation rate of 98.44% after culturing at 32 °C and pH 7 for 24 h. At a nicotine concentration of 8.0 g / L, the TR14 strain achieved a nicotine degradation rate of only 8.01% after culturing at 32 °C and pH 7 for 24 h. This indicates that the strain has high tolerance and significant potential for degrading high concentrations of nicotine. This invention provides an excellent strain for the production of nicotine-degrading microorganisms and has significant application value in the tobacco industry and environmental protection.
[0059] Example 4: Analysis of the ability of strain TR14 to degrade nicotine, protein, and starch in B2F tobacco leaves from the upper part of southern Anhui province.
[0060] Laboratory tobacco fermentation was carried out on representative upper tobacco leaves (B2F) from Anhui Province in 2022 using the selected strain TR14.
[0061] TR14 was streaked onto LB agar plates and incubated at 37 °C for 12 h. A single colony was picked and transferred to 5 mL of liquid LB medium and incubated at 37 °C and 200 rpm for 12 h. The colony was then transferred to 100 mL of liquid LB medium to prepare a seed culture and incubated at 37 °C and 200 rpm for 12 h to obtain the seed culture.
[0062] The prepared seed culture was centrifuged and diluted with sterile water to a cell concentration of 1.0 × 10⁻⁶. 8 After adding CFU / mL, the solution was sprayed evenly onto the surface of the tobacco leaves at a concentration of 20% (w / w) and mixed thoroughly. The control (CK) was uninoculated. The tobacco leaves were cultured in a temperature and humidity controlled incubator at 28 ℃ and 65% relative humidity for 15 days. This resulted in a 18.37% decrease in nicotine content, a 6.71% decrease in protein content, and a 4.9% decrease in starch content. The tobacco leaves also showed a significant reduction in irritation, a smoother smoke, and an increased aroma.
[0063] Example 5: Analysis of the ability of strain TR14 to degrade nicotine, protein, and starch in B3F tobacco leaves from the upper part of southern Anhui province.
[0064] Laboratory tobacco fermentation was carried out on representative upper tobacco leaves (B3F) from Anhui Province in 2022 using the selected strain TR14.
[0065] Centrifuge the seed culture from Example 4, dilute and resuspend in sterile water to a cell concentration of 1.0 × 10⁻⁶. 8 After adding CFU / mL, it was evenly sprayed onto the surface of tobacco leaf B3F at an addition rate of 20% (w / w) and mixed evenly. CK was the control without inoculation. After culturing in a temperature and humidity controlled incubator at 28℃ and 65% relative humidity for 15 days, the nicotine content of tobacco leaves decreased by 39.15%, the protein content decreased by 15.6%, the starch content decreased by 16.6%, the irritation of tobacco leaves was significantly reduced, the smoke was mellow, and the aroma of tobacco was increased.
[0066] Example 6: Inhibitory effect of strain TR14 on tobacco mold mycelium
[0067] The obtained bacterial strains and molds were cultured using the plate confrontation method. A 3 mm bacterial disc was inoculated into the center of PDA medium using a punch. The sieved bacterial strains were streaked and inoculated 2 cm away from the mold. The moldy bacterial strains were inoculated alone as a control. Each group of strains was replicated in 30℃ for 2 days. The width of the inhibition zone was measured and the relative inhibition rate was calculated.
[0068] Relative inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.
[0069] The antagonistic effect of strains in plate confrontation test is as follows Figure 5As shown in the figure. Measurements and calculations revealed that strain TR14 exhibited a relative inhibition rate of 22.01% against *Tobacco Mycorrhiza*, demonstrating a good antagonistic effect.
[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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.
Claims
1. A pseudomonad strain TR14, characterized in that: The Pseudomonas strain TR14 is deposited at the China Center for Type Culture Collection (CCTCC), classified and named Pseudomonas sp. TR14, with a deposit date of November 8, 2024, and accession number CCTCC NO: M 20242492.
2. The application of the pseudomonad strain TR14 according to claim 1 in the degradation of nicotine, characterized in that: The Pseudomonas strain TR14 was inoculated into a nicotine-enriched medium to degrade nicotine. The enrichment medium is formulated as follows: K2HPO4 13.3 g / L, KH2PO4 4 g / L, (NH4)2SO4 0.1 g / L, yeast extract 1.0 g / L, trace element solution 10 mL / L, autoclaved at 121℃ for 20 min, and nicotine added after sterilization by filtration through a 0.22 µm filter membrane, with a nicotine content of 0.5 g / L. The formula for the trace element solution is as follows: Weigh 1.0 g of MgSO4·7H2O, 0.4 g of MnSO4·H2O, 0.2 g of CaCl2·2H2, 0.2 g of CuCl2·2H2O, and 0.02 g of FeSO4·7H2O, dissolve them in 0.1 mol / L HCl solution, and bring the volume to 100 mL. The degradation system was maintained at a temperature of 23℃ to 37℃ and a pH of 6.0 to 9.
0.
3. The application according to claim 2, characterized in that: The inoculum size for Pseudomonas strain TR14 was 1%.
4. The application according to claim 2, characterized in that: The degradation system was set at a temperature of 32°C and a pH of 7.
0.
5. The application according to claim 2, characterized in that: Preparations of nicotine-degrading formulations were prepared using the aforementioned Pseudomonas strain TR14 or its bacterial culture.
6. The application according to claim 5, characterized in that: Using a concentration of 1×10 7 ~1×10 9 Tobacco was sprayed with a bacterial solution of Pseudomonas strain TR14 at CFU / mL.
7. The application according to claim 6, characterized in that: The application rate is 15-25%, and after spraying, the plants are cultured at 28℃ and 65% humidity for 14-16 days.
8. The use of the Pseudomonas strain TR14 according to claim 1 in the preparation of antibacterial agents, characterized in that: The antibacterial agent has a good inhibitory effect on tobacco mold.