Pseudomonas strain TR14 and application thereof in nicotine degradation

Abstract

The invention discloses a pseudomonas strain TR14 and application of the pseudomonas strain TR14 in nicotine degradation, the pseudomonas strain TR-14 for degrading nicotine is separated from soil where tobacco is planted throughout the year, the pseudomonas strain TR-14 has good degradation capacity on nicotine, the degradation rate on 7g / L nicotine is 98.44% after the pseudomonas strain TR14 is cultured for 24 hours, and the highest tolerance reaches 8g / L nicotine. The nicotine content of upper tobacco leaves can be effectively degraded by utilizing the strain.

Description

Technical Field

[0001] The invention relates to a Pseudomonas strain TR14 (Pseudomonassp.TR14) and application of the strain in degrading nicotine, belonging to the technical field of microbial application. Background Art

[0002] Nicotine, also known as nicotine, is the main alkaloid present in tobacco, accounting for more than 95% of the alkaloid content, and is also one of the main harmful components in cigarettes and tobacco leaves. Nicotine is highly toxic and addictive. Adults can be poisoned by taking 4 to 8 mg of nicotine at a time, and children can die by taking 40 to 60 mg at a time. Large doses of nicotine can inhibit the nerve center and cause cardiac paralysis, which can be fatal in severe cases. During the smoking process, nicotine will also be nitrosated to produce strong carcinogens unique to tobacco, such as nitrosamines (TSNA), which cause great harm to the human body. Nicotine not only has side effects such as toxicity and carcinogenesis to humans, but also affects the ecological structure of the soil, pollutes groundwater, causes serious pollution to the soil and environment, and disrupts the ecological balance. Therefore, reducing the content of nicotine in tobacco leaves and the environment is of great significance to maintaining human health and protecting the ecological environment.

[0003] At present, the degradation of nicotine is mainly focused on biological treatment. Compared with physical and chemical treatment methods, microbial degradation of nicotine has the characteristics of strong degradation ability, rich functional microbial species, diverse degradation pathways, and little impact on the environment. Microbial degradation of nicotine is considered to be a more economical and effective treatment method for nicotine degradation. A large number of studies have shown that the use of microorganisms to degrade nicotine has important significance and development prospects in reducing tobacco harm, protecting the environment, and improving the utilization rate of tobacco resources. The Chinese patent application with application number 202310934714.9 discloses a Hunan Pseudomonas MGJ-2 and its application in nicotine degradation, and the deposit number is CGMCC No. 25121. After the nicotine inorganic salt culture medium was treated with strain MGJ-2 for 25 hours, the degradation rate of 500mg / L nicotine was 99.9%. The Chinese patent application with application number 200710070805.3 discloses a new nicotine-degrading bacterium-Pseudomonas ZUTSKD and its application. The bacterium can degrade nicotine, and the maximum nicotine concentration that can be tolerated is 5.5g / L. Chinese patent CN113462611A discloses a nicotine-degrading Arthrobacter urealyticum and its application, but the efficiency of nicotine degradation is low, and 2g / L of nicotine can be completely degraded after 104 hours of culture.

[0004] The above strains have effective performance in degrading nicotine in tobacco leaves, but the nicotine degradation ability of microbial degrading bacteria is not very strong, and the nicotine concentration tolerance is generally low. In addition, some existing bacteria only degrade nicotine and cannot guarantee various quality indicators of tobacco, making it difficult to apply them in actual production. Therefore, it is urgent to cultivate strains that can degrade nicotine more effectively, coordinate tobacco quality, and play a wider application value. Summary of the invention

[0005] At present, the nicotine degradation ability of microbial degradation bacteria is weak, the nicotine tolerance concentration is low, and some bacteria only degrade nicotine, and cannot guarantee various quality indicators of tobacco. In order to solve the above problems, the present invention screens strains that efficiently degrade nicotine from tobacco soil in Anhui Province and prepares them into bacterial agents. The bacterial agents have good universality in reducing nicotine in tobacco leaves, and can have a good nicotine reduction effect on upper tobacco leaves at different grades. At the same time, the strain has strong comprehensive performance, can reduce the protein and starch content of tobacco leaves, and the strain fermentation liquid also has a good inhibitory effect on tobacco leaf mold.

[0006] The Pseudomonas strain TR14 of the present invention is deposited in the China Center for Type Culture Collection, classified and named Pseudomonas sp.TR14, with a deposit date of November 8, 2024, and a deposit number of CCTCC NO: M 20242492. The strain is obtained from the soil of a tobacco field planted all year round in Jing County, Xuancheng City, Anhui Province.

[0007] The method for separating and purifying the Pseudomonas strain TR14 of the present invention comprises the following steps:

[0008] Step 1: Take 5 g of tobacco field soil and add it to a flask containing 100 mL of enrichment medium, and culture it at 28°C and 200 rpm for 48 h; take 1 mL of bacterial suspension and transfer it to another flask containing 100 mL of enrichment medium, and continue to culture under the same conditions; repeat the culture three times to achieve the purpose of enrichment culture;

[0009] Step 2: Dilute the fermentation broth 10 1 -10 4 times, spread it into the screening medium, and culture it at 28°C for 48 h; screen the nicotine-tolerant strains, then inoculate them into LB solid medium for purification, and store the obtained single colonies in glycerol tubes at 4°C.

[0010] The enrichment medium has the following formula: 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, 121°C high pressure steam sterilization for 20 min, nicotine is added after sterilization by filtration with a 0.22 µm filter membrane, and the nicotine content is 0.5 g / L.

[0011] The formula of 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, and dissolve them in 0.1 mol / L HCl solution to make the volume to 100 mL.

[0012] The screening medium was prepared based on the enrichment medium formula, with 1.5% agar powder additionally added, and the nicotine content was adjusted to 1.0 g / L.

[0013] LB medium: 5.0 g / L yeast extract, 10.0 g / L tryptone, 5.0 g / L sodium chloride, pH 7.0. Sterilize with high pressure steam at 121°C for 20 min. Add 1.5% agar to the solid medium.

[0014] The molecular identification steps of the Pseudomonas strain of the present invention are as follows:

[0015] The purified strain was inoculated on LB solid medium and cultured at 28°C for 24 hours. The purified strain was subjected to molecular identification: the TR14 strain 16S rRNA gene sequence was amplified by PCR using bacterial universal primers, and the amplified product was subjected to 1% agarose gel electrophoresis. The target band gel was recovered (about 1500 bp) according to the instructions of the Easy Pure Quick Gel Extraction Kit. At the same time, the TR14 strain 16S rRNA sequence was sequenced on the EzBioCloud website, and the Pseudomonas genus with a high correlation with the sequence was selected for phylogenetic analysis. The phylogenetic evolutionary tree was constructed using the Mega4.0 program.

[0016]

[0017] The invention discloses an application of the Pseudomonas strain TR14 in degrading nicotine.

[0018] Pseudomonas strain TR14 was inoculated with 1% inoculum in an enriched medium containing nicotine (0.5 g / L) and cultured for 24 h. The cells of strain TR14 continued to proliferate within 3-12 h, and the cell concentration decreased during 12-24 h. The maximum OD 600 =1.05767. The maximum degradation capacity of strain TR14 on nicotine reached 100% within 12 h, indicating that the TR14 strain isolated and identified in the present invention has a nicotine degradation effect.

[0019] Furthermore, the temperature of the degradation system is 23°C to 37°C, with the optimum temperature being 32°C; the pH of the degradation system is 6.0 to 9.0, preferably 7.0.

[0020] The Pseudomonas strain TR14 or its bacterial liquid is used to prepare a nicotine-degrading preparation product.

[0021] Furthermore, a concentration of 1×10 7 ~1×10 9 The tobacco was sprayed with a bacterial solution of Pseudomonas strain TR14 at a concentration of CFU / mL.

[0022] Furthermore, the spraying amount was 15-25% (w / w), and the culture was carried out under temperature and humidity controlled conditions of 28°C and 65% humidity for 14-16 days after spraying.

[0023] The invention discloses an application of the Pseudomonas strain TR14 in the preparation of an antibacterial preparation.

[0024] The antibacterial preparation has a good inhibitory effect on tobacco leaf mold.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a nicotine-reducing Pseudomonas TR14 strain, which has high biological activity, is easy to culture, and has stable genetic properties. The present invention shows that the Pseudomonas TR14 strain has a good degradation effect on nicotine at 32°C and pH 7.0. The present invention is used to improve the quality of tobacco raw materials, especially upper tobacco leaves. The process is simple and the reaction conditions are mild. After treatment, the nicotine, protein and starch contents of the tobacco leaves are reduced, the irritation is significantly reduced, the smoke is soft, the tobacco aroma is increased, and the industrial availability of tobacco leaves is significantly improved. Pseudomonas TR14 has a good inhibitory effect on tobacco mold. The TR14 strain can be used as a bacterium of a new microbial preparation to degrade the nicotine content of tobacco, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the phylogenetic tree of TR14.

[0028] Figure 2 This is the comparison and identification result of TR14 bacteria of the present invention.

[0029] Figure 3 is the nicotine degradation ability of strain TR14.

[0030] Figure 4 This graph shows the effect of nicotine concentration on nicotine degradation by the TR14 strain.

[0031] Figure 5 This is the result of strain TR14 antagonizing tobacco leaf mold. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with embodiments.

[0033] The Pseudomonas strain TR14 of the present invention is classified and named Pseudomonas sp.TR14, and is deposited in China Center for Type Culture Collection on November 8, 2024, with a deposit number of CCTCC NO: M 20242492. The strain is obtained from the soil of a tobacco field planted all year round in Jing County, Xuancheng City, Anhui Province.

[0034] Example 1: Isolation and identification of nicotine-degrading microorganisms

[0035] The method for isolating the Pseudomonas strain for nicotine degradation comprises the following steps:

[0036] Prepare the 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 high-pressure steam sterilization at 121°C for 20 min, nicotine was sterilized by filtration with a 0.22 µm filter membrane and then added as needed.

[0038] Trace element solution: weigh 1.0 g MgSO4·7H2O, 0.4 g MnSO4·H2O, 0.2 g CaCl2·2H2, 0.2 g CuCl2·2H2O, and 0.02 g FeSO4·7H2O, and dissolve them in 0.1 mol / L HCl solution to make up to 100 mL.

[0039] Isolation medium: enrichment medium supplemented with 1.5% agar powder.

[0040] LB medium: 5.0 g / L yeast extract, 10.0 g / L tryptone, 5.0 g / L sodium chloride, pH 7.0. Sterilize with high pressure steam at 121°C 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 a flask containing 100 mL (nicotine content of 0.5 g / L) of enrichment medium, and cultured at 28°C, 200 rpm for 48 h. 1 mL of bacterial suspension was transferred to another flask containing 100 mL of enrichment medium, and cultured under the same conditions for 3 times to achieve the purpose of enrichment culture. The fermentation liquid was then diluted and applied to the screening medium (nicotine content of 1.0 g / L), and cultured at 28°C for 48 h. After enrichment and separation, bacteria that can tolerate nicotine were screened, codenamed TR14, and then inoculated on LB solid medium for purification, and the obtained single colonies were stored in glycerol tubes and stored in a refrigerator at 4°C.

[0042] Steps for molecular identification of Pseudomonas strains:

[0043] The purified strain was inoculated onto LB solid medium and cultured at 28°C for 24 h. The purified strain was subjected to molecular identification: the 16S rRNA gene sequence of the TR14 strain was amplified by PCR using bacterial universal primers, and the amplified product was subjected to 1% agarose gel electrophoresis. The target band gel was recovered (about 1500 bp) according to the instructions of the Easy Pure Quick Gel Extraction Kit. At the same time, the 16S rRNA sequence of the TR14 strain was sequenced on the EzBioCloud website, and the Pseudomonas genus with a high correlation with the sequence was selected for phylogenetic analysis. The phylogenetic evolutionary tree was constructed using the Mega4.0 program. The amplified sequence was sequenced, and the similarity of the 16S rRNA gene sequence of the strain was compared in the database. Strains with a similarity higher than 98.65% were considered to be strains of the same genus. The results showed that the TR14 strain belonged to the genus Pseudomonas ( Figure 2 ), phylogenetic tree such as Figure 1 shown.

[0044] The 16S rRNA nucleotide sequence of the strain is as follows:

[0045]

[0046] The Pseudomonas strain is used to degrade nicotine.

[0047] Example 2: Relationship between the degradation rate of nicotine and the growth rate of strain TR14

[0048] The TR14 glycerol bacteria stored at -80℃ were inoculated into fresh liquid LB medium at a ratio of 1:100, activated at 200 rpm and 37℃ for 12h to obtain seed bacteria, and then the seed liquid was inoculated into fresh liquid LB medium at a ratio of 1:100, and cultured at 200 rpm and 37℃ with shaking until the logarithmic growth phase. The bacterial liquid was centrifuged at 12000rpm for 5min to collect the bacteria, resuspended with an equal volume of enrichment medium and centrifuged, washed twice, and resuspended with enrichment medium to obtain degradation seed bacteria TR14.

[0049] The activated and degraded strain TR14 was inoculated with 1% inoculum in a nicotine-containing (0.5 g / L) enrichment medium and cultured at pH 7.0, 32°C, and 200 rpm for 24 h, with the uninoculated nicotine medium as the 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 OD600nm was determined by spectrophotometry. At the same time, the culture fluids of 3 h, 6 h, 9 h, 12 h, 15 h, 18 h, and 24 h were aspirated, centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was taken, and the supernatant was diluted with 0.05 mol / L HCl to an appropriate absorbance range. The changes in the characteristic absorption peak of nicotine at 260 nm were determined using 0.05 mol / L HCl as the reference solution.

[0050] The results are as follows Figure 3 As shown in the figure, the cells of strain TR14 continued to proliferate within 3-12 h, and the cell concentration decreased during 12-24 h. The maximum OD 600 =1.05767. The nicotine concentration in the culture medium decreased significantly after 6 hours, and the maximum degradation capacity of nicotine by strain TR14 reached 100% within 12 hours. This indicates that the TR14 strain isolated and identified in the present invention has a nicotine degradation effect.

[0051] Example 3: Nicotine degradation rate of strain TR14 under different culture conditions such as temperature, pH, and nicotine concentration

[0052] TR14 strain cell suspension (OD 600 1) Inoculate into 30 mL of basal medium for cultivation. Take samples regularly to measure bacterial growth and nicotine degradation. Repeat each experiment 3 times.

[0053] Culture temperature: Nicotine concentration was 0.5 g / L, the temperatures were 23, 28, 32, 37 and 42°C, pH was 7.0, and culture was carried out at 200 rpm.

[0054] Initial pH of the culture medium: The initial pH was 5, 6, 7, 8 and 9, respectively. The nicotine concentration was 0.5 g / L. The culture was carried out at 32°C and 200 rpm.

[0055] Nicotine initial concentration: Nicotine concentrations were 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 g / L, respectively, and cultured at pH 7.0, 32°C, and 200 rpm.

[0056] The rate of nicotine degradation of strain TR14 at 12 h under different culture conditions of temperature, pH, etc. was determined. The results are shown in Table 2. Strain TR14 can grow in the range of 23 ℃~37 ℃ and grows well in the range of 23 ℃~37 ℃. The optimal growth temperature of strain TR14 is 32 ℃. At this temperature, strain TR14 reaches the maximum growth and the highest nicotine degradation rate. The results are shown in Table 3. Strain TR14 grows well in the range of pH6.0~9.0. The optimal pH for cell growth is 7.0. Under this pH condition, strain TR14 reaches the maximum growth and the highest nicotine degradation rate.

[0057]

[0058] The degradation rate of nicotine by TR14 strain under different nicotine concentrations and other culture conditions was determined for 24 h. Figure 4 The results showed that when the nicotine concentration exceeded 7.0 g / L, the TR14 strain degraded nicotine at a slow rate. When the nicotine concentration was 7.0 g / L, the TR14 strain had a nicotine degradation rate of 98.44% after being cultured at 32°C and pH 7 for 24 h. When the nicotine concentration was 8.0 g / L, the TR14 strain had a nicotine degradation rate of 8.01% after being cultured at 32°C and pH 7 for 24 h, indicating that the strain had a high tolerance and had significant potential in degrading high-concentration nicotine. The present invention provides an excellent strain for producing degraded nicotine and has a good 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 upper tobacco leaves in southern Anhui

[0060] The screened strain TR14 was used to carry out laboratory tobacco fermentation on the representative upper tobacco leaves B2F of Anhui in 2022.

[0061] Streak TR14 onto an LB plate and culture at 37 °C for 12 h. Pick a single colony and transfer it to 5 mL of liquid LB medium and culture it at 37 °C, 200 rpm, for 12 h. Transfer it to 100 mL of liquid LB medium as seed solution and culture it at 37 °C, 200 rpm, for 12 h to obtain seed solution.

[0062] The prepared seed solution was centrifuged and diluted with sterile water to resuspend the cells to a concentration of 1.0×10 8 CFU / mL, and then sprayed evenly on the surface of tobacco leaves at an addition rate of 20% (w / w) and mixed evenly. CK was the control without inoculation. It was cultured in a temperature and humidity controlled incubator at 28 ℃ and relative humidity of 65% for 15 d, which could reduce the nicotine content of tobacco leaves by 18.37%, the protein content by 6.71%, and the starch content by 4.9%. The irritation of tobacco leaves was significantly reduced, the smoke was softer, and the aroma of tobacco was increased.

[0063] Example 5: Analysis of the ability of strain TR14 to degrade nicotine, protein and starch in B3F upper tobacco leaves in southern Anhui

[0064] The screened strain TR14 was used to carry out laboratory tobacco fermentation on the representative upper tobacco leaves B3F of Anhui in 2022.

[0065] The seed solution in Example 4 was centrifuged and diluted with sterile water to resuspend the cells to a concentration of 1.0×10 8 CFU / mL, and then sprayed evenly on the surface of tobacco leaves B3F at an addition rate of 20% (w / w) and mixed evenly. CK was the control without inoculation. It was cultured in a temperature and humidity controlled incubator at 28°C and a relative humidity of 65% for 15 days. The nicotine content of tobacco leaves decreased by 39.15%, the protein content decreased by 15.6%, and the starch content decreased by 16.6%. The irritation of tobacco leaves was significantly reduced, the smoke was softer, and the aroma of tobacco was increased.

[0066] Example 6: Inhibitory effect of strain TR14 on tobacco mold hyphae

[0067] The obtained strains were cultured with molds by the plate confrontation method. A 3 mm bacterial sheet was taken with a hole puncher and inoculated in the center of the PDA medium. The screened strains were streaked and inoculated 2 cm away from the molds. The moldy strains were inoculated alone as a control experiment. Each group of strains was replicated 3 times and cultured at 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] Antagonistic effect of strains in plate confrontation test Figure 5As shown. Through measurement and calculation, the relative inhibition rate of strain TR14 against tobacco mold was 22.01%, and the antagonistic effect was good.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Pseudomonas strain TR14, characterized in that: The Pseudomonas strain TR14 was deposited in the China Center for Type Culture Collection, classified and named Pseudomonassp.TR14, with a deposit date of November 8, 2024 and a deposit number of CCTCC NO: M 20242492.

2. Use of the Pseudomonas strain TR14 according to claim 1 in degrading nicotine.

3. The use according to claim 2, characterized in that: Pseudomonas strain TR14 was inoculated into an enriched medium containing nicotine to degrade nicotine.

4. The use according to claim 3, characterized in that: The enrichment medium has the following formula: 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, 121°C high pressure steam sterilization for 20 min, nicotine is added after sterilization by filtration with a 0.22 µm filter membrane, and the nicotine content is 0.5 g / L.

5. The use according to claim 3, characterized in that: The inoculum size of Pseudomonas strain TR14 was 1%.

6. The use according to claim 3, characterized in that: The temperature of the degradation system is 23°C~37°C, and the pH of the degradation system is 6.0~9.

0.

7. The use according to claim 6, characterized in that: The temperature of the degradation system was 32°C, and the pH of the degradation system was 7.

0.

8. The use according to claim 2, characterized in that: The Pseudomonas strain TR14 or its bacterial liquid is used to prepare a nicotine-degrading preparation product.

9. The use according to claim 8, characterized in that: The concentration was 1×10 7 ~1×10 9 The tobacco was sprayed with a bacterial solution of Pseudomonas strain TR14 at a concentration of CFU / mL.

10. The use according to claim 9, characterized in that: The spraying amount is 15-25%. After spraying, the culture is carried out at 28℃ and 65% humidity under controlled temperature and humidity conditions for 14-16 days.

11. Use of the Pseudomonas strain TR14 according to claim 1 in preparing an antibacterial preparation.

12. The use according to claim 11, characterized in that: The antibacterial preparation has a good inhibitory effect on tobacco leaf mold.

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