Pseudomonas and application thereof

Abstract

The invention relates to the technical field of nicotine degradation, in particular to pseudomonas and application thereof.The pseudomonas has high degradation capacity on nicotine, nicotine can serve as the only carbon source and nitrogen source, nicotine can be efficiently degraded at the room temperature (about 25 DEG C), and the degradation efficiency is 92.61%; meanwhile, the invention provides a method for remarkably reducing the nicotine content of the air-cured burley tobacco leaves and improving the taste of the tobacco leaves, and the method comprises the steps that the washed and resuspended bacterial liquid is sprayed on the air-cured burley tobacco leaves, fermentation is conducted for 48-72 h at the temperature of 26-28 DEG C, and then fermentation is terminated at the high temperature. Through the method, the tobacco leaf strength can be reduced, and the taste is improved. The invention establishes a novel method for degrading nicotine and improving the flavor of the burley tobacco leaves by using the specific pseudomonas CGMCC No.31421 fermentation strain, and the method has important application value for improving the quality of the burley tobacco leaves in China.

Description

Technical Field

[0001] The invention relates to the technical field of nicotine degradation, in particular to a strain of Pseudomonas and application thereof. Background Art

[0002] Nicotine gives tobacco a special commodity value and makes it a worldwide cash crop. Burley tobacco originated in the United States and is a dark brown air-cured tobacco. Burley tobacco is named for its milky white stems and veins. This tobacco leaf has a low sugar content (less than 1% soluble total sugar) and a high nicotine content, generally 4% to 5%. It is the main raw material for the production of blended cigarettes and can also be processed into cigars, pipe tobacco and chewing tobacco. Therefore, the low carbon source and high nitrogen source environment of burley tobacco after airing has higher requirements for microorganisms than flue-cured tobacco (about 30% soluble total sugar and about 2% nicotine). In recent years, with the gradual increase in the production of burley tobacco leaves, the nicotine content of some burley tobacco leaves has also gradually increased, especially the upper leaves (up to 9%), resulting in poor compatibility and quality decline of high-nicotine content tobacco leaves. The proportion of use in the high-end cigarette formula of industrial enterprises is not high, resulting in this part of tobacco leaves cannot be fully utilized.

[0003] Based on current research reports, one of the main ways to reduce the nicotine content in tobacco is the regulation of microorganisms and enzymes. This approach is highly efficient and specific, and maintains the original quality of tobacco leaves. Up to now, research on microbial degradation of nicotine in tobacco leaves has increased, but there are relatively few practical applications. The main reasons are: first, the fermentation temperature required for microbial treatment of tobacco leaves is relatively high, requiring a special fermentation room, which increases costs for industrial enterprises; second, the degradation effect of microorganisms directly acting on tobacco leaves is not ideal, especially burley tobacco has a low sugar content, and many microorganisms are difficult to grow normally. Therefore, research on improving tobacco leaf quality through microorganisms and enzyme preparations is mainly focused on flue-cured tobacco and cigars, and there is less research on microorganisms to reduce nicotine in burley tobacco and improve the quality of burley tobacco leaves.

[0004] In response to the current problem of high nicotine content and poor quality of domestic burley tobacco leaves, Summary of the invention

[0005] The invention provides a strain of Pseudomonas and application thereof. The strain of Pseudomonas has the ability to degrade nicotine and can be used to solve the problems of high nicotine content, low tobacco leaf utilization rate and weak flavor existing in current domestic burley tobacco leaves.

[0006] In a first aspect, the present invention provides a strain of Pseudomonas, which is screened from burley tobacco foam after sun-drying. The Pseudomonas strain is from the General Microbiological Center of China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101), with a deposit number of CGMCC No. 31421, a deposit date of July 22, 2024, and a recommended classification name: Pseudomonas sp.

[0007] This strain can be cultured and degrade nicotine at room temperature of 25°C, and can grow normally with nicotine as the only carbon and nitrogen source and reduce 92.61% of nicotine within 24 hours and 93.49% of nicotine within 48 hours. It is particularly suitable for reducing the nicotine content in low-sugar environments such as burley tobacco leaves.

[0008] In a second aspect, the present invention provides use of the Pseudomonas strain and / or a bacterial solution containing the strain in degrading nicotine.

[0009] In a third aspect, the present invention provides use of the Pseudomonas strain and / or a bacterial solution containing the strain in reducing nicotine in tobacco leaves.

[0010] In a fourth aspect, the present invention provides the use of the Pseudomonas strain and / or a bacterial solution containing the strain in enhancing the characteristic flavor of tobacco leaves.

[0011] Preferably, the tobacco leaves in the above application are burley tobacco leaves.

[0012] In a fifth aspect, the present invention provides a nicotine-degrading preparation, characterized in that the preparation comprises the above-mentioned Pseudomonas strain and / or bacterial liquid.

[0013] In a sixth aspect, the present invention provides a method for degrading nicotine or reducing the nicotine content of tobacco to enhance the characteristic flavor of tobacco leaves, specifically, treating a sample at room temperature with the above-mentioned Pseudomonas CGMCC No.31421 strain and / or a bacterial solution containing the strain.

[0014] Preferably, when the method is used to reduce the nicotine content in air-cured burley tobacco leaves and enhance the characteristic flavor of the tobacco leaves, the method comprises the following steps:

[0015] (1) Spraying bacterial solution: spraying the cured burley tobacco leaves with bacterial solution of Pseudomonas sp. CGMCC No. 31421;

[0016] (2) Fermentation treatment: at 26-28°C for 48-72 hours;

[0017] (3) Termination of treatment: The fermentation was terminated by high temperature, and the samples were dried in an oven at 120°C to reduce the moisture content of the tobacco leaves to about 10-14%.

[0018] Preferably, the concentration of the bacterial solution in the above-mentioned bacterial solution spraying step is 0.35×10 7 CFU / ml, the ratio of sprayed bacterial solution to tobacco leaves is 100ml / kg.

[0019] The characteristics of burley tobacco leaves are that the total nicotine (including nicotine, nornicotine, nicotine, and nicotine) content is relatively high, about 5%, and the content is highest in the upper leaves, which can reach 9%, while the total sugar content is low, only 1%, which is relatively unfavorable for the growth of microorganisms. However, the Pseudomonas CGMCC No. 31421 used in the present invention can adapt to this low-carbon source and high-nitrogen source environment and degrade the nicotine therein.

[0020] Although the optimum growth temperature and nicotine degradation temperature of Pseudomonas CGMCC No.31421 in the present invention are 25°C, considering that the tobacco leaf drying oven is close to an open environment, the actual temperature of the bacterial liquid sprayed on the tobacco leaves will be slightly lower than the ambient temperature due to the influence of evaporation and heat absorption. Therefore, an ambient temperature slightly higher than 25°C is used to treat the tobacco leaves. This temperature setting is more suitable for the storage and fermentation stages in actual industrial production.

[0021] The above method can reduce the nicotine content in burley tobacco leaves by up to 28%, and the fermentation temperature is room temperature, the cost is low and the operation is simple, and it can be directly carried out in the storage area / oven.

[0022] In a seventh aspect, the present invention provides a method for improving the taste of finished burley tobacco, which comprises treating tobacco leaves of finished burley tobacco using the above method.

[0023] Preferably, the finished burley tobacco products include blended cigarettes, cigars, pipe tobacco and chewing tobacco.

[0024] The beneficial effects of the present invention include:

[0025] (1) A Pseudomonas strain CGMCC No.31421 that can efficiently degrade nicotine was discovered. This strain can efficiently degrade nicotine under low carbon source conditions.

[0026] (2) Using the above strains to treat burley tobacco leaves can reduce the nicotine content of burley tobacco, reduce the strength of tobacco leaves, improve sensory coordination, increase available tobacco leaf resources, and improve economic benefits.

[0027] (3) Using the above strains to treat tobacco leaves can increase the characteristic aroma of burley tobacco leaves. Through the action of microorganisms, the aroma quality and oral aftertaste of burley tobacco are significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is the 16SDNA comparison analysis result of Pseudomonas CGMCC No.31421 provided by the present invention.

[0030] Figure 2 The present invention provides the bacterial body and colony morphology of Pseudomonas CGMCC No.31421.

[0031] Figure 3 The nicotine degradation efficiency of the Pseudomonas sp. CGMCC No. 31421 provided by the present invention is tested at 25 and 37° C. Note: The other nicotine-reducing strain 2 is the reported strain Arthrobacter sp. D4.

[0032] Figure 4 The invention discloses a sensory evaluation of burley tobacco leaves sprayed with Pseudomonas CGMCC No. 31421. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0034] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0035] Example 1 Cultivation and identification of strains

[0036] The strain was obtained by screening from the tobacco foam of burley tobacco after sun-drying. After isolation and cultivation, it was stored in a -80℃ low-temperature refrigerator.

[0037] (1) Resuscitation of strains

[0038] Aseptically open the glycerol storage tube of the strain stored in a -80℃ low-temperature refrigerator, take an appropriate amount of bacterial solution with an inoculation loop, streak inoculate it on the corresponding culture medium plate LB solid culture medium, and culture it in a constant temperature and humidity chamber at 25℃ for 18h.

[0039] (2) Preparation of primary seed solution

[0040] Pick out the isolated and purified single colony and inoculate it into 3 mL of LB liquid culture medium, and culture it in a constant temperature shaker at 25°C and 200 rpm for 8-10 h to obtain the primary seed solution.

[0041] (3) Preparation of secondary seed solution

[0042] 1 mL of the first-level seed solution (2% (v / v) inoculation amount) was transferred to 50 mL of LB liquid culture medium and cultured in a constant temperature shaker at 25°C and 220 rpm for 4-5 h to obtain the second-level seed solution.

[0043] LB solid medium: Add 1.2-1.5% agar to LB medium.

[0044] TAE electrophoresis buffer: 50x tris-acetate-EDTA (containing 2 M tris acetate and 0.05 M EDTA, pH 8.3).

[0045] Agarose gel: Add 0.8% to 1.2% agarose to 1x TAE running buffer.

[0046] 16S rDNA detection:

[0047] Extract genomic DNA from the bacteria cultured in LB liquid medium, and then use 1% agarose electrophoresis to perform DNA electrophoresis detection. After the DNA reaches the detectable standard, PCR gene amplification is performed, and the PCR upstream and downstream primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3. 0.5μl of PCR template DNA is added to reach an appropriate concentration, the primer concentration is 0.4μM, the dNTP concentration is 0.2mM, the DNA polymerase is 2.5U, the entire reaction volume is 50μL, the denaturation temperature is 94℃, the annealing temperature is 55℃, the extension temperature is 72℃, and the total cycle is 30 times. The length of the PCR product is estimated to be 1.5kb, and it is detected by agarose electrophoresis, brominated acetonitrile staining, and UV Analyzer. The PCR product is sequenced, and the measured 16S rDNA sequence is shown in SEQ ID NO.1.

[0048] The 16S rDNA sequence of the strain of the invention was compared with the typical 16S rDNA sequence in GenBank, and its 16S rDNA sequence was compared with the 16S rDNA of several Pseudomonas strains, and the similarity reached 99%, but they were not completely the same (see Figure 1 ). Combined with the morphological analysis of the strain (see Figure 2 ): The colony morphology is round, milky white, convex lens-shaped, with complete edges and a small colony diameter, so this strain is Pseudomonas.

[0049] Example 2 Preparation of the test medium for nicotine degradation efficiency of Pseudomonas sp. CGMCC No. 31421 at room temperature:

[0050] Nicotine culture medium (per liter): 13.3 g K2HPO4·3H2O, 4 g KH2PO4, 0.2 g MgSO4·7H2O, 0.5 mL trace elements, 1 g nicotine (passed through a 0.22 μm organic film before addition).

[0051] Trace elements: 0.05g CaCl2·2H2O, 0.05g CuCl2·2H2O, 0.008gMnSO4·H2O, 0.004gFeSO4·7H2O, 0.1g ZnSO4·H2O, 0.1gNa2MoO4·2H2O, 0.05g Na2WO4·2H2O (dissolve in 0.1mol / L HCl, sterilize the empty bottle, and pass through an aqueous phase membrane after preparation).

[0052] Nicotine solid medium: Add 1.5% agar to the nicotine medium. Autoclave at 115℃ for 30min.

[0053] Test methods

[0054] (1) Draw a standard curve of nicotine-absorbance

[0055] First, scan 96% nicotine between 200-500nm to obtain the wavelength corresponding to the maximum absorbance value. Use 96% nicotine as the mother solution, and use 0.05mol / L HCl as the solvent to dilute and prepare nicotine standard solutions of 0.005g / L, 0.010g / L, 0.015g / L, 0.020g / L, 0.025g / L, and 0.030g / L, and then record the absorbance at a wavelength of 259nm. Use 0.05mol / L HCl as the reference solution to adjust the instrument to zero. Set the horizontal axis to nicotine concentration and the vertical axis to absorbance. Draw a standard curve of nicotine absorbance and find the functional relationship.

[0056] (2) Degradation rate was measured using an ultraviolet ELISA instrument

[0057] Inoculate the Pseudomonas CGMCC No.31421 strain and the control strain (reported strain Arthrobacter sp.D4) into 5mL LB liquid medium and culture for 10-12h to obtain seed liquid. Take 1mL of seed liquid and add it into 100mL, 1g / L nicotine liquid medium. Culture it in a constant temperature shaker at 20, 25, 37℃ and 200r / min for 24h and 48h respectively. Take 2mL of supernatant and centrifuge it at 10000r / min for 5min. Dilute the supernatant with 0.05mol / L HCl to the absorbance range of the standard curve.

[0058] Use 0.05 mol / L HCl as the reference solution to adjust the instrument to zero, measure the absorbance of the fermentation supernatant sample at 259 nm, calculate the nicotine content by the absorbance, and determine the nicotine degradation rate of the strain. Nicotine degradation rate = ((initial nicotine content - nicotine content in the fermentation broth) / initial nicotine content) × 100% (see Figure 3 ).

[0059] The results showed that Pseudomonas CGMCC No.31421 could effectively degrade nicotine in the culture medium at 25°C, with a 24-hour nicotine degradation rate of 92.61% and a 48-hour nicotine degradation rate of 93.49%. The degradation efficiency at room temperature was significantly higher than that of the control Arthrobacter sp.D4 strain (24-hour nicotine degradation rate of 55.06% and 48-hour nicotine degradation rate of 93.43%). However, the nicotine degradation effect was weaker than that of the control strain at a higher temperature of 37°C, indicating that Pseudomonas CGMCC No.31421 is more suitable for nicotine degradation at room temperature.

[0060] Example 3 A method for improving the quality of air-cured burley tobacco leaves

[0061] The processing steps of the air-cured burley tobacco leaves are as follows, wherein the blank control is an equal amount of physiological saline:

[0062] (1) Spraying bacterial solution: Take the seed solution of Pseudomonas and centrifuge it in a centrifuge at 4°C and 4200 rpm for 5 minutes, and remove the supernatant. Then resuspend the strain with sterile water, centrifuge it again, and remove the supernatant. Resuspend it with physiological saline and centrifuge it for three times, then measure the OD600 of the bacteria and adjust the concentration of the strain sprayed on the sun-dried burley tobacco leaves to 0.35×10 7 CFU / ml, the ratio of sprayed bacterial solution to tobacco leaves is 100ml / kg.

[0063] (2) Processing temperature: 26-28°C

[0064] (3) Sampling time: 48-72 hours.

[0065] (4) Termination of treatment: The fermentation was terminated by high temperature, and the samples were dried in an oven at 120°C to reduce the moisture content of the tobacco leaves to about 12%.

[0066] (5) Nicotine extraction and content detection in tobacco leaves. The detection method refers to the industry standard YC / T 383-2010 "Gas chromatography-mass spectrometry method for the determination of nicotine, nornicotine, anabasine, myosamine and anabasine in tobacco and tobacco products". The results are shown in Table 1.

[0067] (6) The treated burley tobacco leaves were used to roll cigarettes, and the single-material tobacco was smoked. The results are shown in Figure 4 .

[0068] Table 1 Degradation of nicotine by spraying Pseudomonas CGMCC No.31421 on burley tobacco leaves after air-drying

[0069]

[0070]

[0071] After the burley tobacco leaves were treated by the Pseudomonas fermentation process, the aroma quality of the burley tobacco leaves was improved in the sensory evaluation, the fragrance was prominent, the nicotine content was reduced by 11% and 28%, and the overall coordination was good, which significantly improved the quality of the burley tobacco leaves (see Table 1 and Figure 4 )

[0072] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A strain of Pseudomonas, characterized in that: The Pseudomonas species deposit number is CGMCC No.31421.

2. Use of the Pseudomonas strain and / or a bacterial solution containing the strain as claimed in claim 1 in degrading nicotine.

3. Use of the Pseudomonas strain and / or a bacterial solution containing the strain as claimed in claim 1 in reducing the nicotine content in tobacco leaves.

4. Use of the Pseudomonas strain and / or a bacterial solution containing the strain as claimed in claim 1 in improving the characteristic flavor of tobacco leaves.

5. The use according to claim 3 or 4, characterized in that The tobacco leaves are burley tobacco leaves.

6. A preparation, characterized in that The preparation comprises the Pseudomonas strain described in claim 1 and / or a bacterial solution containing the strain.

7. A method for degrading nicotine or reducing the nicotine content of tobacco to enhance the characteristic flavor of tobacco leaves, characterized in that: The sample is treated at room temperature using the Pseudomonas described in claim 1 and / or a bacterial solution containing the strain or the preparation described in claim 6.

8. The method according to claim 7, characterized in that The method is used to reduce the nicotine content in air-cured burley tobacco leaves, comprising the following steps: (1) Spraying bacterial solution: spraying the bacterial solution of Pseudomonas described in claim 1 on air-cured burley tobacco leaves; (2) Fermentation treatment: 48-72 hours at 26-28°C; (3) Termination of treatment: Use high temperature to terminate fermentation and heat the sample in an oven at 120°C to make the moisture content of the tobacco leaves approximately 10-14%.

9. The method according to claim 8, characterized in that The concentration of the bacterial solution in the step of spraying the bacterial solution is 0.35×10 7 CFU / ml, the ratio of sprayed bacterial solution to tobacco leaves is 100 ml / kg.

10. A method for improving the taste of finished burley tobacco, characterized in that: The method of claim 8 is used to treat the tobacco leaves of the burley tobacco finished products, and the burley tobacco finished products include blended cigarettes, cigars, pipe tobacco and chewing tobacco.

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