Glutamicibacter misouriensis, microbial inoculum and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a strain of glutamicibacter mysorens, a microbial agent and application thereof. The present application successfully screens a strain of glutamicibacter mysorens Cas325 which can degrade nicotine efficiently. The strain can grow with nicotine as the sole carbon and nitrogen source. The nicotine content can be reduced by more than 95.59% after the strain is cultured in LB medium containing 1g / L nicotine for 18h. The strain has high efficiency in degrading nicotine, and can preferentially use nicotine as metabolite in the presence of other carbon and nitrogen sources, and has strong nicotine metabolic capacity. In the tobacco plant experiment, the nicotine content of the tobacco plant is reduced by about 18.08% after the strain is sprayed for 12h, and therefore the strain has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Mysore glutamate, a bacterial agent, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Nicotine, chemically known as 1-methyl-2-(3-pyridyl)-tetrahydropyrrolidine, is a highly bioactive pyridine alkaloid widely found in plants of the genus *Nicotiana* in the Solanaceae family. Its molecular structure contains one pyridine ring and one pyrrolidine ring, giving it unique physicochemical properties: on the one hand, nicotine is extremely lipophilic and water-soluble, allowing it to rapidly diffuse through biofilms and accumulate in organisms; on the other hand, its cyclic structure and the stability of its CN bond make it difficult to be photolyzed or hydrolyzed in the natural environment, resulting in a half-life of 20-30 days, and it can even persist for months in anaerobic soils. Furthermore, nicotine pollution is multi-source: in addition to direct release through smoking, tobacco cultivation, processing, and waste disposal are the main pathways of its spread. It is estimated that approximately 3 million tons of tobacco waste (including stems, broken leaves, and processing residues) are generated globally each year, with nicotine content as high as 3%-5%. If these wastes are directly landfilled or left in the open without treatment, nicotine can leach into the soil and groundwater systems with rainwater, posing a long-term threat to urban water bodies and marine ecosystems. Furthermore, nicotine's harm to human health is multi-systemic, damaging the reproductive system and organs such as the lungs, stomach, and liver. At the ecological level, the toxicity of nicotine to non-target organisms has already drawn widespread attention.

[0004] Given the serious threat nicotine poses to human life and the ecological environment, numerous methods for nicotine degradation and remediation have been reported. Since the 1950s, scientists have been screening microorganisms that can effectively degrade nicotine in order to improve the quality of tobacco products and reduce environmental harm. Currently reported nicotine-degrading bacteria mainly include *Pseudomonas*, *Alcaligenes*, and *Gordonia*; therefore, it is necessary to further expand the resource pool of highly efficient nicotine-degrading microorganisms to provide new technical means for nicotine pollution remediation. Summary of the Invention

[0005] To address the shortcomings of the existing technologies mentioned above, the inventors, through long-term practice and exploration, have finally provided a strain of *Mysore glutamate*, its inoculum, and its applications. Specifically, this strain grows rapidly and has strong adaptability to the environment, can utilize nicotine as its sole carbon and nitrogen source for growth, and possesses highly efficient nicotine degradation capabilities. Based on the above research results, this invention is thus completed.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a strain of Glutamicibacter mysorens Cas325, which was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with the biological accession number CGMCC No. 32932.

[0008] A second aspect of the present invention provides a microbial agent containing the Mysore glutamate bacillus or its fermentation product or its metabolite.

[0009] A third aspect of the present invention provides the use of the above-mentioned Mysore glutamate bacillus or the above-mentioned bacterial agent in the degradation of nicotine or the preparation of nicotine degrading agents.

[0010] A fourth aspect of the present invention provides a method for nicotine degradation, comprising applying the above-mentioned Mysore glutamate bacillus or the above-mentioned bacterial agent to tobacco plants, tobacco planting soil, tobacco planting water sources, tobacco products and / or tobacco industrial waste, thereby performing nicotine degradation treatment.

[0011] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0012] The above-mentioned technical solution successfully screened a highly efficient nicotine-degrading strain, *Glutamicibacter mysorens* Cas325. This strain can grow using nicotine as the sole carbon and nitrogen source. After 18 hours of cultivation on LB medium containing 1 g / L nicotine, the nicotine content can be reduced by more than 95.59%, demonstrating high nicotine degradation efficiency. Furthermore, when other carbon and nitrogen sources are available, it preferentially uses nicotine as a metabolite, exhibiting a strong nicotine metabolism capacity. In experiments on tobacco plants, spraying with this strain for 12 hours reduced the nicotine content of tobacco plants by approximately 18.08%, thus demonstrating good practical application value. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 This is a colony morphology diagram of strain Cas325 (Glutamicibacter mysorens) in Example 1 of the present invention.

[0015] Figure 2 This is the phylogenetic tree of Cas325 (Glutamicibacter mysorens) in Embodiment 1 of the present invention.

[0016] Figure 3 The figure shows the nicotine degradation curve of strain Cas325 (Glutamicibacter mysorens) in Example 2 of this invention after being cultured in LB medium containing 1 g / L nicotine. The peak area is the nicotine absorption peak area detected by HPLC (black curve represents 18h, blue curve represents 24h, green curve represents 42h, and cyan curve represents 48h).

[0017] Figure 4 The growth rate of strain Cas325 (Glutamicibacter mysorens) in Example 3 of this invention after 16h to 48h of culturing in LB medium containing 1-5g / L nicotine.

[0018] Figure 5 The peak area of ​​strain Cas325 (Glutamicibacter mysorens) in Example 3 of this invention was detected by liquid chromatography after 16-48 h of culture in LB medium containing 1-5 g / L nicotine. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0021] In a typical embodiment of the present invention, a strain of *Glutamicibacter mysorens* Cas325 is provided. This strain was deposited on December 5, 2024, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with the biodeposit number CGMCC No. 32932. The *Glutamicibacter mysorens* Cas325 of the present invention was isolated from the rhizosphere soil of tobacco.

[0022] In another specific embodiment of the present invention, a bacterial agent is provided, which contains the Mysore glutamate bacillus or its fermentation product or its metabolites.

[0023] The fermentation product referred to in this invention is used to refer to a fermentation product. The corresponding fermentation product can be obtained from the fermentation culture of *Mysore glutamate* Cas325. Depending on the culture method, it can be a solid fermentation product or a liquid fermentation product; the liquid fermentation product can also be called a fermentation broth or culture medium. The fermentation product of this invention contains bacterial cells and their metabolites. In embodiments of this invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is the supernatant, and in this invention, the supernatant contains the metabolites of *Mysore glutamate* Cas325.

[0024] In this invention, the microbial agent may also include excipients conventionally used in the field of microbial agents, which are not specifically limited herein. Correspondingly, the microbial agent can be any known dosage form suitable for microbial agents, including but not limited to powders, emulsions, emulsifiable concentrates, creams, pastes, colloids, granules, microparticles, and oils, etc., which are not specifically limited herein.

[0025] In another specific embodiment of the present invention, the use of the above-mentioned Mysore glutamate bacillus or the above-mentioned bacterial agent in the degradation of nicotine or the preparation of nicotine degrading agents is provided.

[0026] Furthermore, the application of nicotine degradation can specifically include the application of nicotine degradation in tobacco plants, tobacco growing soil, tobacco growing water sources, tobacco products and / or tobacco industry waste.

[0027] The tobacco products mentioned include tobacco leaves, re-dried tobacco leaves, homogenized tobacco leaves, shredded tobacco, cigarettes, cigars, pipes, electronic cigarettes, and heated tobacco products.

[0028] The tobacco industry waste includes, but is not limited to, solid waste such as tobacco stems and tobacco dust, as well as liquid waste (waste liquid) generated during the tobacco industry production process.

[0029] In this invention, the nicotine degrading agent can be used as an environmental modifier, a plant pesticide, and / or a plant fertilizer.

[0030] In this invention, the environmental modifier can be used as a modifier for soil environment and / or water environment.

[0031] In another specific embodiment of the present invention, a method for nicotine degradation is provided, comprising applying the above-mentioned Mysore glutamate bacillus or the above-mentioned bacterial agent to tobacco plants, tobacco planting soil, tobacco planting water source, tobacco products and / or tobacco industrial waste, thereby performing nicotine degradation treatment.

[0032] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0033] Example 1: Isolation and Identification of Strains Cas325

[0034] 1. Method

[0035] 1.1 Sample Collection and Processing: Rhizosphere soil from tobacco plants infected with bacterial wilt in Tongren, Guizhou Province, was collected in August 2022. The root surface soil was separated by ultrasonic vibration (20s × 2 times) and 10 samples were prepared. -1 ~10 -6 Serial dilution of bacterial culture. Take 10 -3 ~10 -6 0.1 mL of diluted bacterial suspension was spread on NA medium and incubated at 28°C for 48 h. Single colonies were screened based on colony morphology, purified three times by three-zone streak, and the strain with the best growth status was selected for identification and further study on its role in degrading nicotine.

[0036] 1.2 Molecular identification:

[0037] DNA was extracted from the strain (by boiling water bath lysis), and 16S rDNA was amplified by PCR (primers 27F / 1492R). The reaction mixture consisted of 2 μL DNA template, 25 μL 2×Taq Master Mix, 1.5 μL primers, and ddH2O to a final volume of 50 μL. The amplification conditions were: 95℃ for 3 min pre-denaturation, 30 cycles (95℃ for 30 s, 58℃ for 30 s, 72℃ for 1.5 min), and 72℃ for 5 min extension.

[0038] PCR products were sequenced (NIO Biotechnology), BLAST similar sequence search and alignment were performed, and MEGA software was used for multi-sequence homology analysis to construct a phylogenetic tree.

[0039] 2. Results

[0040] A bacterial strain was screened and named Cas325. After incubation at 28°C for 24 hours, it formed pale yellow colonies with smooth edges on NA medium. Figure 1 ).

[0041] Based on 16S rDNA sequence comparison and phylogenetic analysis ( Figure 2 The study found that Cas325 (Glutamicibactermysorens) had the highest similarity (99.78%) to Glutamicibacter mysorens LMG 16219. A phylogenetic tree was constructed by combining the 16S rDNA sequences of Cas325 with those of other standard strains in the Glutamicibacaceae family. Figure 2 It was discovered that Cas325 and Mysore glutamate were clustered together, and Cas325 was ultimately identified as Mysore glutamate.

[0042] Example 2: Effect of fermentation time on the nicotine degradation efficiency of Cas325

[0043] 1. Method

[0044] 1.1 Culture conditions: The strain Cas325 was inoculated into LB medium containing 1 g / L nicotine and cultured at 28℃ with shaking at 200 rpm. Samples were taken at 18 h, 24 h, 42 h and 48 h.

[0045] 1.2 Nicotine detection: After centrifugation at 15000 rpm for 2 min, the supernatant was collected, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC (Agilent TC C18 column, mobile phase 40% acetonitrile, column temperature 30℃, flow rate 0.6 mL / min, injection volume 20 μL, detection wavelength 259 nm).

[0046] 2. Results

[0047] Cas325 was cultured in LB medium containing 1 g / L nicotine. Samples were taken at 18 h, 24 h, 42 h, and 48 h, and the nicotine degradation rate was detected by liquid chromatography. The results showed that the degradation rate of Cas325 was 95.59% at 18 h; while the liquid chromatography at 24 h showed that the nicotine was completely degraded. Figure 3 ).

[0048] Example 3: Effect of different nicotine concentrations on the degradation efficiency of Cas325

[0049] 1. Method

[0050] Strains of Cas325 (1% inoculum) were inoculated into LB medium containing 1-5 g / L nicotine and cultured at 28°C and 200 rpm. OD600 and nicotine residue were measured every 4 h (HPLC detection method was the same as in Example 2).

[0051] 2. Results

[0052] Regarding the growth of Cas325 cells, prior to 40 hours, cell growth was inhibited at all nicotine concentrations. Figure 4 Liquid phase results showed that, regarding the degradation efficiency of nicotine by the strain, 1 g / L and 2 g / L nicotine were completely degraded in 20 h and 40 h, respectively; the degradation rate of 3–5 g / L nicotine was slow. Figure 5 ).

[0053] Example 4: The degradation effect of Cas325 on nicotine in tobacco plants

[0054] 1 Method

[0055] 1.1 Preparation and treatment of bacterial culture:

[0056] Strains Cas325 were cultured overnight in LB medium until OD reached. 600 =1.0, the bacterial solution was diluted 10 times with deionized water and sprayed on the tobacco plants after topping (the whole plant was sprayed twice), and the control group was sprayed with water. There were 3 tobacco plants for each treatment.

[0057] 1.2 Sample Testing:

[0058] Four leaves from each plant were taken after 12 hours of treatment, dried at 72℃, ground into powder, and used for later use. 10g of dried tobacco leaf samples were taken from each treatment, and the nicotine content was determined using a Fourier transform near-infrared spectrometer.

[0059] 2. Results

[0060] The nicotine content in the treated tobacco leaves decreased by 18.08%, which was significantly higher than that in the control group, proving that Cas325 can rapidly degrade nicotine in tobacco plants.

[0061] Cas325 16S rRNA gene sequence

[0062]

[0063] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A strain of Mysore glutamate ( Glutamicibacter mysorens Cas325, this strain was deposited at the China General Microbiological Culture Collection Center on December 5, 2024, with the biological accession number CGMCC No. 32932.

2. A microbial agent, characterized in that, It contains Mysore Glutamate Bacillus Cas325 as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The microbial agent includes excipients in the field of microbial agents.

4. The use of Mysore Glutamate Bacillus Cas325 as described in claim 1 or the bacterial agent as described in any one of claims 2-3 in the degradation of nicotine or the preparation of nicotine degrading agents.

5. The application as described in claim 4, characterized in that, The application of nicotine degradation is specifically selected from the application of nicotine degradation in tobacco plants, tobacco growing soil, tobacco growing water sources, tobacco products and / or tobacco industrial waste.

6. The application as described in claim 5, characterized in that, The tobacco products are selected from tobacco leaves, shredded tobacco, cigarettes, cigars, electronic cigarettes, and heated tobacco products.

7. The application as described in claim 5, characterized in that, The tobacco products are selected from re-dried tobacco leaves and homogenized tobacco leaves.

8. The application as described in claim 5, characterized in that, The tobacco industry waste is selected from solid waste such as tobacco stems and tobacco dust, as well as liquid waste generated during the tobacco industry production process.

9. The application as described in claim 4, characterized in that, The nicotine degrading agent is an environmental modifier.

10. The application as described in claim 9, characterized in that, The environmental modifier is a modifier for soil and / or water environments.

11. A method for degrading nicotine, characterized in that, This includes applying the Mysore Glutamate Bacillus Cas325 of claim 1 or the bacterial agent of any one of claims 2-3 to tobacco plants, tobacco growing soil, tobacco growing water sources, tobacco products and / or tobacco industrial waste.

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