Sphingopyxis strain j-6, microbial preparation and application thereof

Abstract

The present application relates to the field of microbial degradation, in particular to a sphingomonas J-6, microbial preparation and application thereof.The sphingomonas J-6 strain provided by the present application can effectively degrade mesomin, and the degradation is embodied in that the concentration of mesomin decreases with the growth of the sphingomonas J-6, and the sphingomonas J-6 can complete the degradation of mesomin within 17 days.It can be seen that the sphingomonas J-6 strain provided by the present application can effectively solve the harm of mesomin, and has great application value for developing products for degrading mesomin.Meanwhile, the strain can also produce 3-pyridine acetic acid, providing a new way for the production of 3-pyridine acetic acid.

Description

Technical Field

[0001] This invention relates to the field of microbial degradation, and in particular to a strain of Sphingosine Box Bacterium J-6, microbial preparations and their applications. Background Technology

[0002] Tobacco alkaloids include nicotine, nornicotine, esmine, pseudoequiline, and neonicotine, whose composition and content directly affect the sensory evaluation quality, smoke characteristics, and safety of tobacco products. Tobacco alkaloids are the main precursors for the formation of tobacco-specific nitrosamines (TSNAs). Nicotine and nornicotine undergo nitrosation reactions to form 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), respectively, which are carcinogenic to humans. Besides nornicotine, esmine is also a precursor to NNN. The molecular formula of esmine is C9H. 10 N2, molecular weight 146.19. Previously, it was thought that mesmin was an alkaloid unique to tobacco, but it has now been found that in addition to being found in nightshade plants, mesmin is also widely distributed in nuts, wheat, corn, rice, and various fruits and vegetables.

[0003] Currently, the microbial degradation of nicotine has been extensively and thoroughly studied. Researchers have explored the mechanisms by which various microorganisms play a role in nicotine degradation and have made some progress in practical applications. However, research on the microbial degradation of mesmin is still insufficient and remains in the preliminary exploratory stage. Microbial strains capable of degrading mesmin are relatively scarce, and the microbial degradation mechanism of mesmin remains unclear. Given the widespread presence of mesmin in the environment and its potential health threats, screening and isolating more microbial strains capable of degrading mesmin from various environments and studying the degradation mechanisms of these strains in depth is particularly crucial and urgent. These studies will help us to better understand the transformation process of mesmin in the environment and within organisms, and will also enhance our scientific understanding of the microbial degradation of tobacco alkaloids. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of *Sphingosine Chaetomium* J-6, a microbial preparation thereof, and its application, in order to solve the problems existing in the prior art. The strain provided by this invention can effectively degrade mesmin and also produce 3-pyridineacetic acid.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a strain of Sphingopyxis sp. J-6, which has the accession number CCTCC NO: M 20241981.

[0007] This invention provides the application of the above-mentioned Sphingosine Box Bacterium J-6 in the preparation of products that degrade mesmin.

[0008] This invention provides a biological agent for degrading mesmin, wherein the active ingredient of the biological agent includes the aforementioned Sphingosine Box Bacterium J-6.

[0009] Preferably, the biological agent further includes excipients.

[0010] This invention provides the application of the above-mentioned Sphingosine Box Bacterium J-6 or the above-mentioned biological agents in the degradation of mesmin.

[0011] This invention provides a method for degrading mesmin, the method comprising the following steps:

[0012] The above-mentioned biological agents are mixed with the substances to be degraded to achieve the effect of degrading mesmin.

[0013] This invention provides the use of the above-mentioned Sphingosine Box Bacterium J-6 in the preparation of products that produce 3-pyridineacetic acid.

[0014] The present invention provides a biological agent for producing 3-pyridineacetic acid, wherein the active ingredient of the biological agent includes the above-mentioned Sphingosine Box Bacterium J-6.

[0015] Preferably, the biological agent further includes excipients.

[0016] The present invention provides the use of the above-mentioned Sphingosine Box Bacterium J-6 or the above-mentioned biological agent in the production of 3-pyridineacetic acid.

[0017] The present invention discloses the following technical effects:

[0018] The *Sphingosine Boxerella* strain J-6 provided by this invention can effectively degrade mesmine. Specifically, the concentration of mesmine decreases as *Sphingosine Boxerella* J-6 grows, and the strain can completely degrade mesmine within 17 days. Therefore, the *Sphingosine Boxerella* strain J-6 provided by this invention can effectively solve the problem of mesmine hazards and has significant application value for developing products that degrade mesmine. Simultaneously, this strain can also produce 3-pyridineacetic acid, providing a new route for the production of 3-pyridineacetic acid. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0020] Figure 1 A phylogenetic tree of strain J-6 constructed based on the 16S rRNA gene sequence;

[0021] Figure 2 The ANI value (%) between strain J-6 and 6 closely related strains;

[0022] Figure 3 This is the standard curve for Maxim's curve;

[0023] Figure 4 The standard curve for 3-pyridineacetic acid;

[0024] Figure 5 The degradation curve of sphingosine mononitrate by *Sphingosine Mononitrate* J-6, the cumulative curve of 3-pyridineacetic acid, and the growth curve of strain J-6 were obtained at a concentration of 500 mg / L. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1: Screening and identification of Sphingopyxis sp. J-6, a sphingo-degrading bacterium.

[0031] Tobacco rhizosphere soil samples were collected from Xiaochengzhuang, Biyang County, Zhumadian City, Henan Province. 5g of soil sample was mixed with 100mL of sterilized liquid mineral salt containing 500mg / L esmine, and 5 sterilized glass beads with a diameter of 4mm were added. The bacterial culture was obtained by shaking and culturing at 30℃ and 160 rpm for 7 days. The liquid mineral salt culture medium consisted of: 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, and trace element solution 1 mL / L. The pH was adjusted to 7.0-7.5, and the medium was then autoclaved (121℃, 30 min). The trace element solution consisted of: 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, and AlCl3·6H2O 0.05 g / L. Next, 5 mL of bacterial culture was inoculated into the same culture medium and cultured for another 7 days. This process was repeated several times to enrich the mysmine-degrading strains.

[0032] The enriched bacterial solution was serially diluted using 0.85% sterile physiological saline, and then the different dilutions (10⁻⁶) were further diluted. -2 -10 -6The bacterial suspension was evenly spread onto R2A plates (R2A medium composition and 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). The spread plates were incubated at 30°C for 7 days. After incubation, single colonies with different morphologies were selected for streak purification. The purified strains were then inoculated into sterilized liquid mineral salt medium containing 500 mg / L of mesmin to conduct a mesmin degradation test, evaluating the mesmin degradation ability of different strains. The ability of a strain to degrade mesmin is determined by regularly observing changes in the color and turbidity of the culture medium. If significant changes in the color or turbidity of the culture medium are observed, it is preliminarily determined that the strain has the ability to degrade mesmin.

[0033]

[0034] The 16S rRNA gene sequence of strain J-6 was compared using the EzBioCloud database (https: / / www.ezbiocloud.net / identify). The comparison results showed that strain J-6 has a high similarity to the genus *Sphingopyxis*, and specifically to *Sphingopyxis panaciterrae* Gsoil 124. T The 16S rRNA sequence showed the highest similarity, reaching 99.50%, and was most similar to Sphingopyxis chilensis S37. T Secondly, it reached 99.29%.

[0035] Based on the comparison results, the 16S rRNA gene sequences of nine type strains with 16S rRNA gene sequences similar to those of strain J-6 were selected. A phylogenetic tree was constructed using MEGA software with the neighbor-joining method. The results are as follows: Figure 1 As shown in the figure, the bootstrap value is set to 1000; "T" indicates the type strain; and the GenBank accession number for the strain's 16S rRNA gene sequence is in parentheses after the strain name. Figure 1 It can be seen that strain J-6 is related to *Sphingopyxispanaciterrae* Gsoil 124. T They clustered together, a result consistent with the 16S rRNA gene sequence alignment.

[0036] Simultaneously, based on the 16S rRNA gene sequence alignment results, six model strains with high 16S rRNA gene sequence similarity to strain J-6 and with complete genome sequences available in public databases were selected. The genome sequences of these six model strains were downloaded, and the ANI values ​​between strain J-6 and the six selected model strains were calculated using OAT software. The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the ANI values ​​of strain J-6 and the 6 strains are 82.26%-94.67%, which are lower than the threshold of 95.0%-96.0% for bacterial species classification.

[0037] Based on the above results, strain J-6 is a novel species of the genus *Sphingopyxis*, distinct from all previously described species. It was named *Sphingopyxis* sp. J-6 and was biodeposited. This strain was deposited on September 14, 2024, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20241981, at Wuhan University, Wuhan, China.

[0038] Example 2: Determination of the ability of Sphingosine Box Bacterium J-6 (strain J-6) to degrade mesmin and produce 3-pyridineacetic acid

[0039] In this embodiment, we quantified the cell growth of strain J-6 by measuring the absorbance of the culture medium at a wavelength of 600 nm.

[0040] To determine the content of mesmin and 3-pyridineacetic acid, we used high-performance liquid chromatography (HPLC). A standard curve for mesmin was plotted, as shown below. Figure 3 As shown, the standard curve equation is y = 20980.47x - 511856.7, R0 2 = 0.999847, where y is the peak area and x is the amount of mesmin. Plot the standard curve of 3-pyridineacetic acid, as shown below. Figure 4 As shown, the standard curve equation is y = 13770.1x - 233912.7, R0 2 =0.998968, where y is the peak area and x is the 3-pyridineacetic acid content. Standard curves for mesmin and 3-pyridineacetic acid were used to calculate the content of mesmin and 3-pyridineacetic acid in the culture medium. The detection conditions for high-performance liquid chromatography (HPLC) were as follows: a Waters XSelect HSS T3 column (4.6 × 250 mm, 5 μm), a mobile phase of methanol:KH₂PO₄ (20 mmol / L, pH = 2.6) = 3:97 (v / v), a flow rate of 1.0 mL / min, a column temperature of 30℃, an injection volume of 10 μL, and a detection wavelength of 268 nm. Quantification was performed using the external standard method based on peak area.

[0041] The experimental design included two treatment groups: an experimental group and a control group. The experimental group consisted of 1 mL of OD... 600 J-6 bacterial suspension with a pH adjusted to 0.6 was inoculated into liquid mineral salt medium containing mesmin (mesmin concentration of 500 mg / L) and cultured in a shaker at 30℃ and 160 rpm; the control group was uninoculated liquid mineral salt medium containing mesmin (mesmin concentration was the same as the experimental group).

[0042] Samples were taken at regular intervals, centrifuged at 4℃ and 12000 rpm for 2 min, and the supernatant was collected and filtered twice through a 0.22 μm aqueous filter membrane. Finally, high-performance liquid chromatography (HPLC) was used to detect the levels of mesmin and 3-pyridineacetic acid. The degradation curves of mesmin, the accumulation curve of 3-pyridineacetic acid, and the growth curve of strain J-6 at a concentration of 500 mg / L mesmin are shown below. Figure 5The results showed that the concentration of sphingomyelin decreased with the growth of *Sphingosine Boxerella* J-6, and *Sphingosine Boxerella* J-6 could completely degrade sphingomyelin within 17 days. Simultaneously with the degradation of sphingomyelin, 3-pyridineacetic acid gradually accumulated, indicating that *Sphingosine Boxerella* J-6 can utilize sphingomyelin to produce 3-pyridineacetic acid.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Sphingosine Box Bacteria ( Sphingopyxis sp.) J-6, characterized in that, The preservation number of the sphingosine box bacteria J-6 is CCTCC NO: M 20241981.

2. The use of the Sphingosine Trichophyton J-6 as described in claim 1 in the preparation of products that degrade mesmin.

3. A biological agent for degrading mesmin, characterized in that, The biological agent includes the Sphingosine Box Bacterium J-6 as described in claim 1.

4. The biological agent according to claim 3, characterized in that, The biological agent also includes excipients.

5. The use of the Sphingosine Boxer J-6 as described in claim 1 or the biological agent as described in claim 3 or 4 in the degradation of mesmin.

6. A method for degrading mesmin, characterized in that, The method includes the following steps: The biological agent of claim 3 or 4 is mixed with the substance to be degraded; the substance to be degraded contains mesmin.

7. The use of the Sphingosine Trichophyton J-6 of claim 1 in the preparation of a product that produces 3-pyridineacetic acid.

8. A biological agent for producing 3-pyridineacetic acid, characterized in that, The biological agent includes the Sphingosine Box Bacterium J-6 as described in claim 1.

9. The biological agent according to claim 8, characterized in that, The biological agent also includes excipients.

10. The use of the Sphingosine Boxer J-6 of claim 1 or the biological agent of claim 8 or 9 in the production of 3-pyridineacetic acid.

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