CN113773980A - Enterobacter nicotianae NLB1 for degrading nicotine and application thereof - Google Patents

Abstract

The invention discloses an enterobacter nicotianae NLB1 for degrading nicotine and application thereof, wherein a new bacterial strain for degrading nicotine, namely enterobacter nicotianae NLB1, is obtained by separation, screening and identification, and is preserved in Guangdong province microbial culture collection GDMCC at 3-18 months in 2021, the bacterial strain number is GDMCC NO: 61569. the nicotine content can be reduced by using the strain; the degradation process is efficient and safe, and the strain has good application prospect in the tobacco industry and environmental protection.

Description

Enterobacter nicotianae NLB1 for degrading nicotine and application thereof

Technical Field

The invention relates to the technical field of nicotine degradation, in particular to enterobacter nicotianae NLB1 for degrading nicotine and application thereof.

Background

Tobacco is one of the main economic crops in China, and the tobacco is the first crop in the world regardless of area or total yield. Although tobacco is a favorite crop with both yield and quality, cigarettes are consumed by smoking, the quality is particularly important, and the yield is improved on the basis of ensuring the quality. Tobacco contains a plurality of harmful substances, most of which are nicotine and tar, which seriously harm human health.

Nicotine (Nicotine) is a main component in tobacco alkaloid, is used as the specific alkaloid of tobacco plants, and accounts for 90-95% of the total alkaloid of tobacco cultivars. Accounting for 0.05-6.0 percent of the dry weight of the tobacco. Nicotine is a main index for evaluating the quality of tobacco leaves and is also a main index for measuring the taste of tobacco products. The content of nicotine in tobacco leaves is generally required to be between 1.5 and 3.5 percent, preferably about 2.5 percent. The nicotine content is too high, the physiological strength is high, the taste is deteriorated, the irritation is enhanced, the spicy irritation of smoke is increased, the taste is influenced, the safety is poor, and the health is damaged; the nicotine content is too low, the taste is flat, and the requirement of smoking cannot be met. In the process of burning and smoking tobacco leaves, nicotine is nitrited to generate the special strong carcinogen N-nitrosamine in the tobacco leaves. Therefore, the maintenance of a proper amount of stable nicotine content in the tobacco leaves is very important for ensuring the cigarette quality and maintaining the health of consumers. Therefore, the cultivation of a good variety with proper nicotine content becomes a problem which needs to be solved urgently in sustainable development of the tobacco industry.

As more nitrogen fertilizer is used in the current tobacco planting process, the nicotine content in the tobacco exceeds the normal level. At present, the nicotine content of flue-cured tobacco in China is higher, the nicotine content reaches 3% -4%, and the nicotine content of burley tobacco is even as high as 6%. At present, more traditional means such as timely topping, breeding low-nicotine variety planting, proper fertilization and the like are adopted at home, so that the nicotine content can be reduced, the operation is complicated, the cost is high, the labor intensity is high, and the effect is low, and therefore the traditional means are not suitable for effectively degrading the excessively high nicotine content in the current tobacco. How to reduce the nicotine content in tobacco leaves is a practical problem faced by various cigarette enterprises.

The beneficial microorganisms and the metabolites thereof are utilized to reduce the nicotine content, so that the method is efficient and safe, and does not have adverse effect on the main quality of cigarettes, thereby being a potential method. In 1947, Enders et al (Enders C, Windisch S. the decomposition of nicotine by yeast [ J ]. biochem.,1947,318:54-62) began a nicotine decomposition study using yeast, and found that yeast could not completely degrade nicotine. In the same year, the results of experiments on industrial fermentation treatment of Tobacco by using culture solutions of Deharyomyces nicotinae and Micrococcus nicotinae, which are prepared by mixing the above two bacterial solutions, show that the two bacterial solutions can improve the flavor and aroma of Tobacco and reduce the nicotine content of Tobacco by 0.45 percent and 0.83 percent respectively, and the nicotine content of Tobacco treated by mixing the two bacterial solutions is reduced by 0.61 percent. The nicotine solution was treated with Pseudomonas (Pseudomonas) isolated from the soil for 24 hours by Newton et al (Newton, Richard P, Geiss, et a.l Process for the reduction of nicotine content: USA,4037609.1977-07-26), resulting in a substantial reduction of the nicotine content and a pH of the solution from 6.4 to 4.6. However, the decomposition of nicotine was stopped due to the decrease of pH and the increase of the number of colonies, and about 35% of nicotine was not degraded. In 1957, Frankenburg et al (Frankenburg W G, Vaitekunas AA. chemical studios on nicotine degradation by micro organic chemical derivatives from the surface of the tobacco seeds [ J ]. Coresta,1957,1:0034.) not only degraded 5% -20% of nicotine, but also accelerated other oxidation reactions of the tobacco leaves, improved the taste quality of the tobacco, further research found that the degradation of nicotine in the process of drying cigar leaves was mainly degraded in the initial stage of drying; in 1978, Lawrence et al (Lawrence E, Gravely, Louisville K Y, Vernon L.Geiss, et al. Process for the reduction of nicotine and nicotine content of tobaco by microbial tobacco [ P ]. US Patent:4557280,1978) used Pseudomonas (Pseudomonas putida purified) to degrade nicotine in tobacco, and found that the nicotine content of mixed cut tobacco (1: 1) of burley tobacco and flue-cured tobacco was reduced from 2.0% to 0.85% on average and that the nicotine content of each cigarette was reduced from 1.58mg to 0.98mg by smoke analysis. At the same time, the company also screened both cellulomonas and pseudomonas bacteria. Treating 20.4kg of stemmed tobacco leaves with 37.2kg of bacterial liquid, keeping the water content between 68 and 70 percent, and drying the tobacco leaves to 14.5 percent after the treatment. After the treatment, the nicotine content of the tobacco leaves is reduced from 3.5 percent to 1.65 percent.

Microorganisms which are found in tobacco plants, soil and aged tobacco leaves and which are capable of degrading nicotine are primarily the species Pseudomonas (Pseudomonas putid), Cellulomonas sp, Arthrobacter nicotianae (Arthrobacter nicotinoides), Arthrobacter globiformis (Arthrobacter globiformis), Arthrobacter nicotinovorans (Arthrobacter nicotinovorans), Arthrobacter oxydans (Arthrobacter oxydans), Alcaligenes dispergerium (Alcaligenes paradoxus) and Nicotiana plomginifolia. Among them, Arthrobacter nicotinovorans (Arthrobacter nicotinovorans) and Pseudomonas pulida (Pseudomonas pulid) are excellent strains capable of degrading nicotine. Many foreign nicotine-degrading bacteria have been successfully applied to practical production, but are mainly used for nicotine degradation after tobacco fermentation and processing. In China, the initial stage of searching microorganisms capable of effectively degrading nicotine is still in the field, deep research on the molecular mechanism of nicotine degradation by microorganisms is still lacked, and especially, research on regulating nicotine synthesis and degrading nicotine by microorganisms in the process of tobacco cultivation is still blank. Therefore, the application of the nicotine-degrading bacteria to the field degradation of high-concentration nicotine in tobacco leaves is a field worth of intensive research in China.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides an enterobacter nicotianae NLB1 for degrading nicotine and application thereof.

The first purpose of the invention is to provide an enterobacter nicotianae NLB1 for degrading nicotine.

The second purpose of the invention is to provide the application of the Enterobacter nicotianae NLB1 in the degradation of nicotine.

It is a third object of the present invention to provide a method for reducing nicotine in tobacco.

It is a fourth object of the present invention to provide a formulation for degrading nicotine.

In order to achieve the purpose, the invention is realized by the following scheme:

the Enterobacter tabaci (Enterobacter tabaci) NLB1 from the female imago of the brown planthopper is preserved in 18 months 3 in 2021 in Guangdong province culture Collection center, and the preservation number is GDMCC NO: 61569. the method utilizes a culture medium with nicotine as a unique carbon source, 16S rRNA and physiological and biochemical indexes to separate and identify the strain NLB 1; meanwhile, the degradation capability of the strain NLB1 on nicotine under different pH values, temperatures and tolerance is measured. The result shows that the strain is enterobacter nicotianae NLB1, the optimum pH value and the temperature of the strain NLB1 for degrading nicotine maximally are respectively 30 ℃, and the pH value is 7.0; the NLB1 seed bacteria are inoculated into a nicotine liquid culture medium at 5% for culturing for 72 h. The maximum degradation amount is reached after 36h, and the concentration of the strain NLB1 is about 10⁹cfu/mL, maximum nicotine degradation capacity of 50%.

Accordingly, the present invention claims a nicotine-degrading Enterobacter nicotianae (Enterobacter tabaci) NLB1, which was deposited at 18/3/2021 in the culture collection of microorganisms and cell cultures (GDMCC) of guangddong province, with the culture collection number GDMCC NO: 61569.

the invention also claims the application of the Enterobacter nicotianae NLB1 in the degradation of nicotine.

The invention also claims a method for reducing nicotine in tobacco, and the nicotine is degraded by using the Enterobacter tabacum NLB 1.

Preferably, the Enterobacter nicotianae NLB1 degrades nicotine at 25-40 ℃.

More preferably, the Enterobacter nicotianae NLB1 degrades nicotine at 30-40 ℃.

Further preferably, said Enterobacter nicotianae NLB1 degrades nicotine at 30 ℃.

Preferably, the Enterobacter nicotianae NLB1 degrades nicotine at pH 6.0-8.0.

More preferably, said enterobacter nicotianae NLB1 degrades nicotine at pH 7.0.

Most preferably, said Enterobacter nicotianae NLB1 degrades nicotine at 30 ℃ pH 7.0.

Preferably, the Enterobacter nicotianae NLB1 degrades nicotine by culturing in an inorganic salt basal medium.

Preferably, in a concentration of 1X 10⁸～1×10¹⁰cfu/mL of the bacterial liquid of the Enterobacter nicotianae NLB1 of claim 1.

More preferably, in a concentration of 1X 10⁹cfu/mL of the bacterial liquid of the enterobacter nicotianae NLB 1.

The invention also claims a preparation for degrading nicotine, which contains the Enterobacter nicotianae NLB 1.

The invention also claims the application of the Enterobacter Nicotiana NLB1 in the preparation of products for degrading nicotine.

Compared with the prior art, the invention has the following beneficial effects:

the patent separates, screens and identifies a new bacterial strain of the microbe for degrading the nicotine, namely the Enterobacter tabacum NLB1, and the bacterial strain can reduce the nicotine content; the degradation process is efficient and safe, and the strain has good application prospect in the tobacco industry and environmental protection.

Drawings

FIG. 1 shows the plate of NLB1 isolate.

FIG. 2 is a phylogenetic analysis of Enterobacter Nicotiana NLB 116S rDNA of Nilaparvata lugens. Note: labeled on each branch: GenBank serial No. + strain name.

FIG. 3 shows the state of the E.niloticus NLB1 in nicotine medium.

FIG. 4 shows the nicotine degrading ability of Nicotiana nilotica NLB 1.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 isolation culture and identification of Enterobacter Nicotiana NLB1 of Nilaparvata lugens

Isolation culture of nicotine-resistant Nilaparvata lugens Enterobacter Nicotiana NLB1

1. Preparation of Selective isolation Medium

LB culture medium: 10g peptone, 5g yeast extract, 10g NaCl, 15g agar, dissolved in 1L sterile ddH₂And adjusting the pH to 7.0 in water.

EB medium: 3g of beef extract powder, 10g of tryptone, 15g of yeast powder, 5g of sodium chloride, 10g of glucose and 15g of agar are dissolved in 1L of sterile water, and the pH value is adjusted to 7.2.

NA medium: 10g of tryptone, 3g of beef extract, 5g of sodium chloride and 15g of agar are dissolved in 1L of sterile water, and the pH value is adjusted to 7.0.

2. Bioassay and sampling

Selecting rice growing for 15 days and having uniform growth vigor, and dividing the rice into two groups, wherein each group is repeated by three, and each group is repeated by 4-6 plants.

Adopting a rice seedling dipping method to carry out brown planthopper bioassay: pulling out the rice with roots, washing the rice with clear water, airing the rice in a shady and cool place until the surface is anhydrous, soaking the rice in a solution with the nicotine concentration of 160mg/L and the clear water for 1min, taking the rice out of the shady and cool place until the surface is anhydrous, wrapping the root of the rice with wet cotton, placing the rice in a raw measuring cup, inoculating 15 adult brown planthopper female adults within 1-2 d, placing the rice in an artificial climate box for culturing for 3d, and then respectively sucking all groups of the living brown planthoppers. Climate box conditions: temperature: 28 ℃; humidity: 85 percent; the photoperiod: 14-10 h.

3. Grinding and coating plate

The collected planthoppers were sterilized on their surfaces (30 s by 70% ethanol, 30s by 2% sodium hypochlorite, and 1min by shaking twice in clear water) and transferred into test tubes containing 200 μ L PBS, ground into homogenate by a hand-held electric grinder, centrifuged slightly, and the supernatant was collected. Diluting with 5 concentration gradients according to dilution 10^-1，10^-2，10^-3，10^-4，10^-5After doubling, the cells were plated on three selective separation media for culture, and each treatment was repeated three times. Culturing in a 37 deg.C constant temperature incubator, and observing every 24 h.

4. Continuous purification culture

After single fungus grows out in the selective separation culture medium. Firstly, single colonies are picked according to the color, size and shape of the colonies and are continuously streaked and purified on a corresponding culture medium for more than 5 times, and then streaked plates for separating strains are photographed, and the result is shown in figure 1. Transferring the monoclone into corresponding liquid culture medium, shaking bacteria until the bacteria exponential growth phase, storing in 25% glycerol water solution, and freezing at-80 deg.C in refrigerator for use.

II, identification of Enterobacter Nicotiana NLB1

An enterobacter nicotianae from the brown planthopper female imago is identified and obtained from the obtained strains, is named as NLB1, and further strain identification is carried out.

1. Conventional biological assays

(1) Morphological characteristics of bacterial colony

In terms of morphology, the NLB1 strain forms a pale translucent wet colony on an LB plate, which is circular and has a uniform edge, and is often rod-shaped because gram staining is not easily discolored. The bacteria are rod-shaped and gram-negative bacteria under microscopic observation. The bacteria have facultative aerobe, the optimal growth temperature is 30 deg.c, the nutrient requirement is not high, and the bacteria grow well in common culture medium LB.

(2) Physiological and biochemical characterization of tobacco bacillus NLB1

The results of the physiological and biochemical characteristics of NLB1 obtained by the invention are shown in Table 1: can decompose beta-galactosidase, arginine diazyme, ornithine decarboxylation, citric acid, glucose, mannitol, sorbitol, sucrose, honey pool, amygdalin and arabinose. The pH range of normal growth is 6-8, and the growth temperature is 25-40 ℃.

TABLE 1 physio-biochemical characteristics of NLB1

2. 16S rDNA identification

(1) The stored genomic DNA of the NLB1 strain was extracted using a bacterial genomic DNA extraction Kit (TIANAmp Bacteria DNA Kit) from Tiangen organisms, and 16S rDNA of the bacterium was amplified using the extracted DNA as a template and 16S rDNA universal primers 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers. After the PCR system is gently mixed, the mixture is centrifuged for a short time and placed on a PCR instrument according to the following steps: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 50 ℃ for 15s, and extension at 72 ℃ for 15s for 30 cycles; 72 ℃ for 5 min; storing at 10 deg.C and 4 deg.C, detecting PCR product with 1% agarose gel, cutting, recovering and purifying, and sequencing and identifying by Guangzhou Ongke biotechnology.

(2) Phylogenetic analysis of Enterobacter Nicotiana NLB1

Bacterial genome amplification analysis was performed with bacterial 16S universal primers 27F and 1492R, and SeqMan (DNAStar) splice sequence to obtain amplification product with nucleotide sequence as SEQ ID NO:1, which was then blast aligned with rRNA/ITS database in NCBI. The near-edge sequence of NLB1 strain is aligned and analyzed by ClustalW software, then a phylogenetic tree is constructed by adopting an adjacent method (Neighbor-Joining) by Mega7.0 software, the bootstrap value is adjusted, and the reliability of the phylogenetic tree is checked. The results are shown in FIG. 2, and show that NLB1 has the highest similarity with Enterobacter tabacum strain YIM Hb-3(NR 146667.2:13-1450), indicating that NLB1 is of the genus Enterobacter tabacum.

The Enterobacter tabacum (Enterobacter tabaci) NLB1 was deposited at 18 th 3/2021 in the Guangdong province culture Collection center with the deposit number GDMCC NO: 61569, the preservation address is No. 100 Michelia furiosa of Guangzhou, Guangdong province.

Example 2 cultivation of Enterobacter Nicotiana NLB1 on Nicotine Medium

First, experiment method

1. Culture medium

Inorganic salt basal medium (MSM): 13.3g K₂H PO₄、4.0g KH₂ PO₄、0.1g(NH₄)₂SO₄Yeast powder 1.0g, trace elements 10.0mL (1.0g MgSO)₄·7H₂O、0.4g MnSO₄·H₂O，0.2g CaCl₂·2H₂O、0.2g CuCl₂·2H₂ O、0.02g FeSO₄·7H ₂O, dissolved in 0.1 mol/L HCl to 100mL), and distilled water was added to 1L.

Nicotine culture medium: 90% nicotine was filtered through a 0.22 μm filter and added to the sterilized inorganic salt basal medium.

2. Bacterial colony culture

The activated Enterobacter tabacum NLB1 (Enterobacter tabaci) obtained in example 1 was plated on MSM solid medium containing 200mg/L nicotine, and cultured in an inverted state at 30 ℃ for 24 hours in an incubator, and the growth of colonies in the petri dish was observed.

Second, experimental results

The colony growth state is shown in FIG. 3. As is clear from FIG. 3, Enterobacter tabacum (Enterobacter tabaci) NLB1 obtained in example 1 showed a single colony formed on 200mg/L MSM solid supplemented with nicotine, and the colony was clear. It is demonstrated that the Enterobacter tabacum NLB1 obtained in example 1 can grow on a medium containing nicotine and has a nicotine degrading effect.

Example 3 effects of temperature and pH on the Nicotine degradation Rate of NLB1 and the growth Rate of NLB1

Preparation of degradable seed bacteria

Glycerol bacteria (Enterobacter tabacum (Enterobacter tabaci) NLB1 obtained in example 1) stored at-80 ℃ were inoculated at a ratio of 1: 100, inoculating fresh liquid LB culture medium, shaking and culturing at 220rpm/min and 37 ℃ for 14h, and then adding 1: 100 percent of the culture medium is transferred into a fresh LB liquid culture medium again, the OD6 is cultured by shaking at the temperature of 37 ℃ at 220rpm/min₀₀The cells were collected, resuspended in an equal volume of sterile MSM basal salt medium, washed twice by centrifugation, and resuspended in basal salt medium to obtain the degraded seed bacteria.

Second, measuring nicotine concentration by spectrophotometry

Preparing the pure nicotine into working solution with the mass concentration of 5.0g/L by using 0.05mol/L HCl solution, respectively taking 0.01 mL, 0.02 mL, 0.03 mL, 0.04 mL, 0.05 mL and 0.06mL of the working solution, and fixing the volume to 10mL by using 0.05mol/L HCl solution. And measuring the optical density value of the nicotine solution at 260nm under different nicotine mass concentrations by taking 0.05mol/L HCl solution as a reference. The results show that: when the mass concentration of nicotine is 0.005-0.030 g/L, the regression equation is that Y is 35.429X +0.0053, and R2 is 0.9993. It is stated that within this range, the absorbance (Y) is directly related to the nicotine concentration (X), and can be used to reflect the nicotine concentration.

Third, the effect of temperature on growth of strain NLB1 and on nicotine degradation

Inoculating the prepared degrading seed bacteria into basic culture medium MSM containing nicotine (1g/L) according to the inoculation amount of 5%, performing shake culture at 25 deg.C, 30 deg.C, 35 deg.C and 40 deg.C, and culturing at 180r/min for 24 hr, and measuring OD of bacteria by spectrophotometry₆₀₀Absorbance and OD at wavelength₂₆₀The absorbance of nicotine.

Results Table 2 shows that the strain NLB1 can grow in the range of 25-40 deg.C, and grows well in the range of 30-40 deg.C with little difference. The optimum growth temperature of the Enterobacter tabaci (Enterobacter tabaci) NLB1 strain was 30 ℃, at which the maximum growth amount of the Enterobacter tabaci (Enterobacter tabaci) NLB1 strain obtained in example 1 was 0.998 and the nicotine degradation rate was 24.9% at the maximum.

Fourth, the Effect of pH on growth of Strain NLB1 and on Nicotine degradation

Basic culture media MSM containing nicotine (1g/L) are prepared into basic culture media MSM with different pH values, namely 6.0, 6.5, 7.0 and 8.0 basic culture media MSM respectively. Inoculating the seed degrading bacteria into basic culture medium MSM containing nicotine (1g/L) at different initial pH according to 5%, shake culturing at 30 deg.C and 180r/min for 24 hr, sucking 24 hr culture solution, centrifuging at 4 deg.C and 12000rpm for 10min, collecting supernatant, filtering with 0.22 μm water phase filter membrane, diluting the supernatant with 0.05mol/L HCl to 0.005-0.030 g/L, taking 0.05mol/L HCl as reference solution, and determining by OD spectrophotometry₆₀₀Lower sum wavelength OD₂₆₀Further obtaining the growth state of the cells and the degradation condition of the nicotine.

Results Table 2 shows that Enterobacter tabacum (Enterobacter tabaci) NLB1 strain grows well in the pH range of 6.0-8.0. The optimum pH value for cell growth is 7.0, under the condition of the pH value, the maximum growth amount of the Enterobacter tabacum (Enterobacter tabaci) NLB1 strain is 0.998, and the degradation rate of nicotine is the highest and reaches 49.8%.

TABLE 2 NLB1 growth at different pH and different temperature and degradation rate of nicotine

Example 4 Nicotine-degrading ability of Strain NLB1

Preparation of degradation seed bacteria

Glycerol bacteria (Enterobacter tabacum (Enterobacter tabaci) NLB1 obtained in example 1) stored at-80 ℃ were inoculated at a ratio of 1: 100 into fresh liquid LB culture medium, 220rpm, 37 ℃ shaking culture for 14h, then 1: 100 are transferred again into fresh LB liquid medium at 220rpmmin, shaking culture at 37 deg.C OD6₀₀The cells were collected, resuspended in an equal volume of sterile MSM basal salt medium, washed twice by centrifugation, and resuspended in basal salt medium to obtain the degraded seed bacterium NLB 1.

Second, degradation ability of NLB1 to nicotine

The degraded seed bacterium NLB1 was inoculated into 1g/L nicotine culture medium MSM at an inoculum size of 5%, and cultured at 30 deg.C and 180rpm with non-inoculated nicotine culture medium as control. Absorbing culture solution for 6h, 12h, 24h, 36h, 48h and 72h, centrifuging at 12000rpm for 10min at 4 ℃, taking supernatant, filtering with 0.22 mu m water-phase filter membrane, diluting the supernatant with 0.05mol/L HCl to 0.005-0.030 g/L, taking 0.05mol/L HCl as reference solution, and respectively measuring wavelength OD by spectrophotometry₆₀₀And wavelength OD₂₆₀Further obtaining the growth state of the cells and the degradation condition of the nicotine.

Third, experimental results

The seed-degrading bacterium of Enterobacter nicotianae (Enterobacter tabaci) NLB1 obtained in example 1 was inoculated into a nicotine liquid medium at pH 7.0 and 30 ℃ and cultured for 72 hours. The absorbance was measured for 6h, 12h, 24h, 36h, 48h, and 72h, respectively. As shown in FIG. 4, the cell proliferation of the strain NLB1 continued within 0-36 h, the cell concentration decreased within 36-72 h, and the maximum concentration was about 10 within 3d of culture time⁹cfu/mL; the nicotine concentration in the culture medium is obviously reduced after 24h, and the nicotine concentration curve tends to be flat at 60 h. Thus, the ability of strain NLB1 to degrade nicotine under this experimental condition was about 72h, 50%.

Example 5 Strain NLB1 tolerance concentration test

First, experiment method

The Enterobacter tabacum (Enterobacter tabaci) NLB1 degraded seed strain obtained in example 1 was cultured in a common medium to late logarithmic phase, inoculated into a basal medium at an inoculum size of 5%, nicotine with different concentrations was added as a carbon source, NLB1 was cultured at 30 ℃ and 180rpm, sampling was performed for 36 hours, centrifugation was performed at 4 ℃ and 12000rpm for 10min, the supernatant was taken, passed through a 0.22 μm aqueous membrane filter, the supernatant was diluted with HCl at a concentration of 0.05mol/L to a concentration of 0.005-0.030 g/L, and the concentration was 0.05mol/L HCl as reference solution, and spectrophotometry to determine wavelength OD₆₀₀And wavelength OD₂₆₀Further obtaining the growth state of the cells and the degradation condition of the nicotine.

Second, experimental results

The results show that the Enterobacter tabacum (Enterobacter tabaci) NLB1 strain obtained in example 1 can degrade nicotine to 50% when the concentration of nicotine is less than 2g/L, and the growth of cells is enhanced with the increase of the nicotine concentration, and the maximum OD value is reached at 2 g/L. When the nicotine concentration is higher than 2g/L, the growth of cells and the degradation of nicotine are in a downward trend. When the nicotine concentration reaches 6g/L, the Enterobacter tabacum (Enterobacter tabaci) NLB1 strain obtained in example 1 can not grow basically, the substrate effect is obvious, and the degradation of nicotine is stopped.

Example 6 sensory evaluation of tobacco treated with Strain NLB1

First, experiment method

The Enterobacter nicotianae (Enterobacter tabaci) NLB1 strain obtained in example 1 was used for degrading the B2F grade flue-cured tobacco in Yunnan province of 2019 by nicotine treatment, which is specifically as follows: the bacterial liquid of degrading seed bacterium NLB1 is prepared at the concentration of 1 × 10⁹Evenly spraying cfu/mL on redried tobacco strips, turning over tobacco leaves gently, balancing water and standing for 30 minutes; after the moisture is balanced, the tobacco leaves are placed in a shady and cool place indoors and are shaded for 3 days. After the treatment is finished, the tobacco leaves are cut into threads and dried, the moisture of the tobacco threads is controlled to be 12% -12.5%, sensory quality evaluation is carried out, and samples in the same batch which are not treated are used as reference.

Wherein the sensory quality score is (aroma quality × 0.35+ aroma amount × 0.25+ miscellaneous odor × 0.1+ pungency × 0.15+ aftertaste × 0.15) × 11.11.

Second, experimental results

The results show in table 3, after the strain NLB1 is treated, style characteristic indexes of 2019 Yunnan B2F can be obviously improved, smoke concentration and strength are properly reduced, smoke comfort is improved, and formula applicability of tobacco leaves is improved. The NLB1 strain treatment has no obvious influence on the quality characteristic indexes of 2019 Yunnan B2F, and the original quality characteristics of tobacco leaves can be maintained.

Table 3 NLB1 nicotine degradation treatment of redried tobacco grade B2F from Yunnan province of 2019

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

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Claims (10)

1. An Enterobacter nicotianae (Enterobacter tabaci) NLB1 for degrading nicotine, wherein the strain is deposited at 18/3/2021 in GDMCC (China Guangdong province culture Collection center) with the strain deposition number being GDMCC NO: 61569.

2. use of enterobacter nicotianae NLB1 of claim 1 in the degradation of nicotine.

3. A method of reducing nicotine in tobacco, comprising degrading nicotine using enterobacter nicotianae NLB1 of claim 1.

4. The method according to claim 3, wherein the Enterobacter nicotianae NLB1 of claim 1 degrades nicotine at 25 ℃ to 40 ℃.

5. The method according to claim 3, wherein the Enterobacter nicotianae NLB1 of claim 1 degrades nicotine at a pH of 6.0 to 8.0.

6. The method of claim 3, wherein the Enterobacter nicotianae NLB1 of claim 1 is cultured in an inorganic salt basal medium to degrade nicotine.

7. The method of claim 3, wherein the concentration is 1 x 10⁸～1×10¹⁰cfu/mL of the bacterial liquid of the Enterobacter nicotianae NLB1 of claim 1.

8. The method of claim 3, wherein the concentration is 1 x 10⁹cfu/mL of the bacterial liquid of the enterobacter nicotianae NLB 1.

9. A preparation for degrading nicotine, comprising Enterobacter Nicotiana NLB1 according to claim 1.

10. Use of enterobacter nicotianae NLB1 as claimed in claim 1 in the preparation of nicotine-degrading products.

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