A bacterial strain degrading nornicotine, products and uses thereof

Abstract

The present application relates to a kind of degradation bacterial strain of nornicotine, product and its application, the bacterial strain is SMGY-1XX, the strain is identified as Mycolicibacterium sp., is preserved in China typical culture preservation center, and the preservation name is Mycolicibacterium sp. SMGY-1XX, and the preservation number is CCTCC M 2023418.The degradation bacterial strain of nornicotine of the present application can effectively degrade nornicotine, and has great application value for solving the harm of nornicotine, development nornicotine degradation product.

Description

Technical Field

[0001] This invention relates to a nicotine-degrading bacterial strain, product, and its application, belonging to the field of microbial degradation technology. Background Technology

[0002] There are four main alkaloids in tobacco: nicotine, nornicotine, neonicotine, and pseudoequisetine. Nornicotine, also known as demethylnicotine, has the molecular formula C9H. 12 N2, compared to nicotine, is simply the absence of a methyl group on the pyrrole ring. Tobacco alkaloids are the main precursors for the formation of tobacco-specific nitrosamines (TSNAs). Nicotine, nornicotine, neonicotine, and pseudoequiline undergo nitrosation reactions to form 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), respectively. N -Nitronic acid ( N′ -nitrosonornicotine, NNN), N -Nonnicotinic acid ( N′ -nitrosoanatabine (NAT) and N -N-nitrosopyridine ( N′ Nitrosamines (NAB) are four tobacco-specific nitrosamines. Among these, NNK (formed from nicotine) and NNN (formed from nornicotine) are carcinogenic to humans. During tobacco cultivation and cigarette manufacturing, a large amount of tobacco waste is generated annually. This waste contains high concentrations of nicotine and nornicotine, among other tobacco alkaloids. Improper disposal of this waste can lead to the release of harmful alkaloids such as nicotine and nornicotine into the soil and water bodies, polluting the environment, disrupting the ecological balance, and posing a threat to human health.

[0003] Utilizing microorganisms to degrade harmful residues in the ecological environment is of significant value, offering advantages such as low cost, high efficiency, environmental friendliness, and sustainability. Currently, the microbial degradation of nicotine has been studied extensively. However, research on the microbial degradation of nicotine is less developed and still in its early stages. The microbial degradation mechanism of nicotine remains unclear, and there is a shortage of isolated nicotine-degrading strains. Therefore, it is necessary to screen for more nicotine-degrading bacteria from the environment. Summary of the Invention

[0004] The present invention aims to provide a bacterium, product and application for degrading nicotine, to solve the problem of lack of nicotine-degrading strains in the prior art.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A nicotine-degrading bacterial strain, characterized in that the bacterial strain is SMGY-1XX, which has been identified as... Mycolicibacterium sp., deposited at the China Center for Type Culture Collection, under the accession name […]. Mycolicibacterium sp. SMGY-1XX, accession number CCTCCNO: M 2023418, accession date March 28, 2023, accession address Wuhan University, Wuhan, China.

[0006] This invention also provides a product for degrading nicotine, including the nicotine-degrading bacterial strain described in this invention.

[0007] The application of the nicotine-degrading bacterial strain of the present invention in the preparation of nicotine-degrading products.

[0008] The present invention also provides a method for degrading nicotine, which involves using the nicotine-degrading bacterial strain described in the present invention to treat articles containing nicotine.

[0009] Furthermore, preferably, adding nicotinic acid-degrading bacterial strains to a liquid mineral salt culture medium containing nicotinic acid for cultivation.

[0010] Furthermore, preferably, the culture conditions are 25-35℃ and 150-200 rpm.

[0011] The nicotine-degrading bacterial strain of the present invention can effectively degrade nicotine, and has significant application value for solving the harm of nicotine and developing products that degrade nicotine. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 A phylogenetic tree of strain SMGY-1XX constructed based on the 16S rRNA nucleotide sequence; Figure 2 The ANI values ​​(%) of strain SMGY-1XX and 8 closely related strains are given. Figure 3 The standard curve for nicotine reduction; Figure 4The degradation and growth curves of strain SMGY-1XX under 500 mg / L nornicotine conditions are shown. Implementation

[0014] The technical solutions of this embodiment will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0015] (1) Culture medium: Preparation of liquid mineral salt culture medium: K₂HPO₄ 1.5 g / L, KH₂PO₄ 0.5 g / L, MgSO₄ 0.2 g / L, (NH₄)₂SO₄ 1.0 g / L, NaCl 1.0 g / L, trace element solution 1 mL / L, pH adjusted to 7.0-7.5, and autoclaved (121℃, 30 min). The trace element solution formula is: MnSO₄·H₂O 0.13 g / L, ZnCl₂ 0.23 g / L, CuSO₄·H₂O 0.03 g / L, CoCl₂·6H₂O 0.42 g / L, Na₂MoO₄·2H₂O 0.15 g / L, AlCl₃·6H₂O 0.05 g / L.

[0016] R2A medium preparation: tryptone 0.25 g / L, acid-hydrolyzed casein 0.5 g / L, yeast extract 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate 0.1 g / L, sodium pyruvate 0.3 g / L, agar 12.0 g / L, peptone 0.25 g / L, glucose 0.5 g / L, pH adjusted to 7.2.

[0017] Preparation of enrichment medium: Add nornicotinic acid to liquid mineral salt medium to achieve a final concentration of 500 mg / L.

[0018] (2) Screening, isolation and purification of strains Five g of sediment samples collected from rivers in Zhengzhou city were placed in a sterilized Erlenmeyer flask containing 100 mL of enrichment medium. The flask was then incubated on a shaker at 30°C and 160 rpm for 7 days. Next, 5 mL of the bacterial culture was inoculated into 100 mL of fresh enrichment medium and incubated for another 7 days. This process was repeated multiple times to enrich the nicotinic acid-degrading bacteria.

[0019] Take 0.1 ml of the enrichment solution and serially dilute it with 0.85% sterile physiological saline in a 2 mL centrifuge tube. Take 10 ml of the enrichment solution and serially dilute it with 0.85% sterile physiological saline. -2 -10 -6 0.1 mL of each diluted enriched bacterial solution was evenly spread onto R2A plates. The plates were incubated at 30°C, and the growth of the bacteria was observed periodically. After 7 days of incubation, single colonies with different morphologies were picked and further isolated and purified using the streak plate method. The purified strains were inoculated into liquid mineral salt medium containing nicotinic acid to test the ability of different strains to degrade nicotinic acid. Changes in the culture medium were observed periodically; strains showing significant changes in color and turbidity were preliminarily considered to have the ability to degrade nicotinic acid. Finally, it was found that only strain SMGY-1XX had the ability to degrade nicotinic acid.

[0020] (3) Whole genome sequencing and identification of the strain The strain SMGY-1XX was sent to BGI Genomics for DNA extraction and whole-genome sequencing. The whole-genome sequencing employed a combination of second-generation sequencing technology (DNBSEQ platform) and third-generation sequencing technology (Nonopore platform). Sequencing results showed that the SMGY-1XX genome contains only one circular chromosome, with a length of 7,267,506 bp and an average GC content of 66.9%. The 16S rRNA nucleotide sequence of the SMGY-1XX genome was extracted and is shown in SEQ ID NO:1.

[0021] The 16S rRNA nucleotide sequence of strain SMGY-1XX was uploaded to the EZBioCloud database (https: / / www.ezbiocloud.net / identify) for sequence alignment. The alignment results were processed using the "Valid names only" option to filter out non-pattern strains. The alignment results showed that strain SMGY-1XX was similar to... Mycolicibacterium The genus has a high degree of similarity, and Mycolicibacterium conceptionense CCUG 50187 T The 16S rRNA sequence showed the highest similarity, reaching 99.17%, and was consistent with... Mycolicibacterium farcinogenes DSM 43637 T Secondly, it reached 99.10%.

[0022] Based on the comparison results, a phylogenetic tree was further constructed using MEGA software and the neighbor-joining method to compare the 16S rRNA nucleotide sequence of strain SMGY-1XX with the 16S rRNA nucleotide sequences of 12 similar type strains. The results are as follows. Figure 1As shown in the figure: This phylogenetic tree was constructed using the Neighbor-joining method, with a bootstrap value set to 1000; "T" indicates the type strain; the brackets following the strain name contain the accession number (GenBank accession number JGI Project Id) of the strain's 16S rRNA nucleotide sequence.

[0023] Depend on Figure 1 It can be seen that strain SMGY-1XX in Mycolicibacterium This constitutes an independent branch within the genus.

[0024] Based on the 16S rRNA nucleotide sequence alignment results, eight model strains with high 16S rRNA nucleotide sequence similarity to strain SMGY-1XX were selected. The genome sequences of these eight strains were downloaded from the NCBI Assembly database (https: / / www.ncbi.nlm.nih.gov / assembly / ). Then, the ANI (average nucleotide identity) value between strain SMGY-1XX and the selected eight strains was calculated using OAT software. The results are as follows: Figure 2 As shown.

[0025] Depend on Figure 2 It can be seen that the ANI values ​​of strain SMGY-1XX and all 8 strains are 75.70%-76.49%, all below 90%, far below the bacterial species classification threshold of 95.0%-96.0%. Combining the phylogenetic tree results based on 16S rRNA sequences and the ANI analysis results, strain SMGY-1XX is... Mycolicibacterium A new fungal species, distinct from all existing described species, was named... Mycolicibacterium sp. SMGY-1XX.

[0026] The bacterial growth was expressed by measuring the absorbance of the culture medium at 600 nm. Nicotine residue was detected using high-performance liquid chromatography (HPLC), and the nicotine content in the test culture medium was calculated based on the nicotine standard curve. The HPLC detection conditions were as follows: a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm), and a mobile phase of methanol:water = (5:95). v / v The flow rate was 0.6 mL / min, the column temperature was 30℃, the injection volume was 10 μL, the detection wavelength was 259 nm, and the external standard method was used for quantification based on peak area.

[0027] Nicotine reduction standard curve as follows Figure 3 As shown, the equation of the standard curve is y = 36825.6x + 50899.68, R0 2=0.997524, where y is the peak area and x is the nicotine content.

[0028] Add 1 mL OD 600 The strain SMGY-1XX, with a pH adjusted to 0.6, was inoculated into a liquid mineral salt medium containing nicotine (nicotine concentration of 500 mg / L) and cultured in a shaker at 30℃ and 160 rpm. This was the experimental group. The control group consisted of uninoculated liquid mineral salt medium containing nicotine (nicotine concentration consistent with the experimental group). Samples were taken regularly to continuously measure the changes in nicotine concentration during degradation and the growth of the SMGY-1XX strain.

[0029] (2) Determination of nicotine concentration and bacterial growth 2 ml of culture medium at 24 h, 48 h, 54 h, 60 h, 66 h, and 69 h was collected in centrifuge tubes and centrifuged at 12000 rpm for 10 min at 4 ℃. The supernatant was collected, and the mixture was centrifuged again under the same conditions. The supernatant was then filtered twice through a 0.22 μm aqueous filter membrane. Finally, the nicotinic acid content was determined by high-performance liquid chromatography (HPLC). Cell growth was expressed by measuring the absorbance of the culture medium at 600 nm at 24 h, 48 h, 54 h, 60 h, 66 h, and 69 h. The degradation and growth curves of the strain under 500 mg / L nicotinic acid conditions are shown below. Figure 4 .

[0030] The results above show that the concentration of nornicotinic acid decreased with the growth of strain SMGY-1XX, and strain SMGY-1XX has a good degradation effect on nornicotinic acid, and can completely degrade nornicotinic acid within 69 h.

[0031] Although embodiments of the present invention have been described above, any modifications and substitutions made by those skilled in the art without departing from the principles and spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A strain of nicotine-degrading bacteria, characterized in that, The bacterial strain was identified as SMGY-1XX. Mycolicibacterium sp., deposited at the China Center for Type Culture Collection, under the accession name […]. Mycolicibacterium sp. SMGY-1XX, accession number CCTCCNO: M 2023418, accession date March 28, 2023.

2. A product for degrading nicotine, characterized in that: Including the nicotinic bacteria strain described in claim 1.

3. The use of the nicotinic acid-degrading bacterial strain according to claim 1 in the preparation of nicotinic acid-degrading products.

4. A method for degrading nicotine, characterized in that: The nicotine-degrading bacterial strain described in claim 1 or 2 is used to degrade articles containing nicotine.

5. The method for degrading nicotine according to claim 4, characterized in that: Nicotine-degrading bacterial strains were added to a liquid mineral salt culture medium containing nicotine and cultured.

6. The method for degrading nicotine according to claim 5, characterized in that: The culture conditions are 25-35℃ and 150-200 rpm.

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