Serratia marcescens sourced siderophore compound as well as extraction method and application thereof
By screening and preparing the iron carrier Serratiochelin E of Serratiocerene F2-2, the problem of insufficient types of Serratiocerene compounds was solved, and a new drug development with anti-inflammatory activity was achieved.
Patent Information
- Application Number
- CN202510288932.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, there are fewer types of iron carrier compounds of Serratia, which limits its application potential in the fields of medicine, agriculture and environmental restoration.
By screening and culturing Serratiochelin F2-2, the new iron carrier Serratiochelin E was prepared by fermentation and purification methods, including static culture, ultrasonic soaking, solid phase extraction and semi-preparation liquid phase separation, and the Serratiochelin E with a clear structure was obtained.
Serratiochelin E demonstrates good anti-inflammatory activity, with the potential to develop novel anti-inflammatory drugs, which may reduce side effects and improve efficacy.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to a siderophore compound derived from Serratia marcescens, and an extraction method and application thereof. Background Art
[0002] Siderophores (also called siderochromes) are a class of low-molecular-weight compounds secreted by microorganisms under iron-limiting conditions. They can efficiently chelate Fe³⁺ ions in the environment and transport them into the cell through a specific receptor-mediated pathway for microbial metabolism and utilization. This mechanism is not only an important survival strategy for microorganisms to adapt to iron stress environments, but also their unique metal chelating ability and biological activity have shown great potential in the fields of medicine, agriculture, and environmental remediation.
[0003] Serratia marcescens ( Serratia marcescens ) is a Gram-negative, motile, non-spore-forming, rod-shaped γ-proteobacteria. This bacterium has a strong ability to produce secondary metabolites, and common metabolites include prodigiosin, serrawettin, etc. Research shows that Serratia marcescens siderophores not only participate in the virulence regulation of pathogenic bacteria, but also have biological functions such as antibacterial, antioxidant, and regulation of host immune responses, providing important resources for the development of new antibacterial agents, anticancer drugs, and biological control agents. In addition, bacteria of the genus Serratia can also produce a small amount of serratiochelin siderophores, and only 4 such substances have been reported so far. Therefore, isolating and identifying iron-carrying compounds from Serratia marcescens can enrich the types and functions of active products of Serratia marcescens and develop new siderophores. Summary of the Invention
[0004] The purpose of the present invention is to provide a siderophore compound derived from Serratia marcescens, and an extraction method and application thereof.
[0005] To achieve the above purpose, the present invention adopts the following technical solutions: A strain of Serratia marcescens F2-2, whose taxonomic name is Serratia. Marcescens , was deposited at the Guangdong Provincial Microbial Culture Collection Center on February 6, 2025, with the deposit number GDMCC No.65860.
[0006] The present invention also provides the application of the above-mentioned Serratia marcescens F2-2 in the fermentation preparation of the siderophore compound Serratiochelin E, and the structural formula of the Serratiochelin E is as follows: .
[0007] A method for preparing the siderophore compound Serratiochelin E, comprising the following steps: (1) Inoculating the activated Serratia marcescens F2-2 into an SDA liquid medium and culturing to obtain a seed fermentation broth; (2) Inoculating the seed fermentation broth obtained in step (1) into a rice medium and statically culturing at room temperature for 4 weeks to obtain the F2-2 rice ferment; (3) Adding an extraction solvent to the F2-2 rice ferment obtained in step (2), soaking overnight and then mashing, obtaining an extract by two ultrasonic soaking extraction methods, and obtaining a dark red extract after concentration under reduced pressure; (4) Dissolving the extract obtained in step (3), loading it onto a normal-phase solid-phase extraction column, collecting the fractions eluted with petroleum ether-acetone, loading it onto a reversed-phase solid-phase extraction column, eluting with a methanol-water system, and subjecting the eluate to semi-preparative liquid separation after concentration under reduced pressure to obtain Serratiochelin E; The structural formula of the said Serratiochelin E is: .
[0008] Further, the conditions for the culturing in step (1) are culturing in a constant temperature shaking incubator at 28 °C and 150 r / min for 7 days.
[0009] Further, the preparation method of the rice medium in step (1) is soaking 180 g of rice in 240 mL of distilled water overnight and sterilizing at 121 °C for 20 min.
[0010] Further, the extraction solvent for the soaking extraction method in step (3) is ethyl acetate-methanol, the solid-liquid ratio is 1:6; the extraction time is 30 min; the volume ratio of the ethyl acetate-methanol is 85:15.
[0011] Further, the conditions for the semi-preparative liquid separation in step (4) are: the chromatographic column is Reprosil Gold 120C 18 , the mobile phase is isocratic elution with 55% methanol-water, the flow rate is 1.5 mL / min, the injection volume is 100 μL, the detection wavelength is 210 nm, and the retention time is 45 min.
[0012] The present invention also provides the application of the prepared Serratiochelin E in the preparation of anti-inflammatory drugs.
[0013] The beneficial effects of the present invention are as follows: Serratiochelin E belongs to the members of the new serratiochelin. The structure and anti-inflammatory activity of this compound can provide new ideas and lead compounds for the research and development of anti-inflammatory drugs. This compound may exert anti-inflammatory activity by regulating the ferroptosis pathway, and it is expected to develop new anti-inflammatory drugs with fewer side effects and better efficacy. Description of the Drawings
[0015] Figure 1 It is the colony morphology of Serratia marcescens F2-2.
[0016] Figure 2 It is the key HMBC of serratiochelin E and 1 H- 1 H COSY signals.
[0017] Figure 3 It is the high-resolution mass spectrum of serratiochelin E.
[0018] Figure 4 It is the nuclear magnetic resonance hydrogen spectrum of serratiochelin E.
[0019] Figure 5 It is the nuclear magnetic resonance carbon spectrum of serratiochelin E.
[0020] Figure 6 It is the DEPT diagram of serratiochelin E.
[0021] Figure 7 It is the 1 H- 1 H COSY diagram.
[0022] Figure 8 It is the HMQC diagram of serratiochelin E.
[0023] Figure 9 It is the HMBC diagram of serratiochelin E.
[0024] Figure 10 It is the cytotoxicity of serratiochelin E against RAW 264.7 mouse mononuclear macrophages.
[0025] Figure 11 It is the anti-inflammatory activity of serratiochelin E (compared with the normal group, ****P<0.0001). Detailed Implementation Modes
[0026] To make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the scope of the present invention is not limited. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The materials in the following embodiments can be obtained from the market unless otherwise specified.
[0027] Example 1 Screening of Serratia marcescens F2-2: Serratia marcescens ( Serratia. marcescens F2-2) is a bacterium isolated from the silt of the mangrove forest in Zhangjiang Estuary, Fujian. The isolation method is as follows: Take the silt sample, add an appropriate amount of sterile normal saline, shake well and let stand, take the suspension, and dilute it to 10 −2 、10 −3 、10 −4 at three different dilution degrees. Respectively suck 100 μL of the sample from each dilution degree, and use a sterile spreader to evenly spread it on the 216E medium. Incubate it upside down at 28 °C for 2-3 d. After various forms of colonies grow on the plate, use an inoculation needle to pick the target single colony and inoculate it into the 2216E medium (Qingdao Haibo Biotechnology Co., Ltd., batch number: 20240808) by the plate streaking method. After culturing in an incubator at 28 °C for 3-5 d, observe the colony morphology, pick the single colony mycelium, and repeat the above operations until a pure single colony is obtained, numbered F2-2 ( Figure 1 ). Send the single colony plate to Meiji Biotechnology Co., Ltd. for sequencing. Using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492-R (5′ -GGTTACCTTGTTACGACTT-3′), perform PCR amplification and sequencing on the 16S rDNA of the isolated and purified strain. Compare the 16S sequence of F2-2 in the NCBI database by BLAST, select and download several sequences with the highest homology, and finally identify the strain as Serratia marcescens ( S. Marcescens ).
[0028] The present invention screened and obtained a strain of Serratia marcescens ( Serratia marcescens ) F2-2, deposited in Guangdong Provincial Microbiological Culture Collection Center on February 6, 2025, with the deposit number GDMCC No.65860, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0029] Example 2 Bacterial fermentation, extract and monomer acquisition 1. Seed Solution Preparation Under sterile conditions, the activated F2-2 strain SDA plate was taken out, and the agar block containing a single colony was picked out and put into 100 mL SDA liquid culture medium (Guangdong Huankai Microbiological Technology Co., Ltd., batch number: 230413A20), and cultured in a constant temperature shaking incubator at 28°C and 150 r / min for 7 days to obtain the F2-2 seed solution.
[0030] 2. Fermentation of Bacteria and Rice Culture Medium A 1000 mL Erlenmeyer flask was used as a fermentation container to prepare a rice solid culture medium. The culture medium formula for each bottle was 180 g of rice and 240 mL of distilled water. The culture medium was soaked overnight and sterilized at 121°C for 20 min to obtain the rice culture medium. After cooling thoroughly, 10 mL of F2-2 seed solution was transferred with a pipette under sterile conditions and inoculated into the rice culture medium in the fermentation bottle. After sealing, the fermentation bottle was placed at room temperature for static culture for 4 weeks to obtain the F2-2 rice fermentation product.
[0031] 3. Extract Preparation 6 times the amount of ethyl acetate-methanol (85:15) was added to the F2-2 rice fermentation product, which was soaked overnight and crushed. Ultrasonic extraction was used for extraction for 30 min and filtered to obtain a dark red filtrate. 6 times the amount of extraction solvent (ethyl acetate-methanol (85:15)) was added to the residue and extracted once using the same method. The two extracts were combined and concentrated under reduced pressure at 50°C using a rotary evaporator to obtain a dark red extract.
[0032] 4. Isolation of serratiochelin E Take 2 g of the extract obtained in step 3, dissolve it with a small amount of methanol: acetone (1:1, v / v), load it into a normal phase solid phase extraction column (filler is silica gel, 200-300 mesh), let it stand at room temperature for 24 h until the solvent evaporates, use a petroleum ether-acetone system gradient (100%-0%) to elute, collect the target compound fractions, evaporate to dryness and load it into a reverse phase solid phase extraction column (filler is ODS, 50 μm), elute with a methanol - water system (30% methanol, 50% methanol, 70% methanol, 100% methanol), take the 70% eluate, concentrate it under reduced pressure at 50 °C, and separate the concentrated solution by semi - preparative liquid phase to obtain the compound (10 mg) with a yield of 0.5%. The separation conditions are: the chromatographic column is Reprosil Gold 120 C 18 (5 μm, 250 mm, ID: 10 mm), the mobile phase is isocratic elution with 55% methanol - water, the flow rate is 1.5 mL / min, the injection volume is 100 μL, the detection wavelength is 210 nm, and the retention time is 45 min.
[0033] Example 3 Structural Identification of Serratiochelin E The (-) ESI high - resolution mass spectrum of serratiochelin E ( Figure 3 ) showed a quasi - molecular ion peak of 458.1594 for [M - H] - , suggesting that the molecular formula of the compound is C 22 H 25 N3O8, with 12 degrees of unsaturation. The 1 H - NMR showed 6 aromatic proton signals at 6.7~7.4 ppm (Table 1, δ H ), Figure 4 ), 13 The C - NMR spectrum δ C showed 12 aromatic carbon signals in the range of 116.3~150.0 ppm (Table 1, Figure 5 ), indicating that there may be two benzene rings in its structure; the compound also showed three carbonyl carbon signals: δ C 171.4, 168.9, 165.0 (Table 1). Based on this inference, the compound should have another ring structure, and its structure was determined by 2D NMR experiments. Figure 2 Key HMBC and 1 H - 1 H COSY signals for serratiochelin E. In the 1 H - 1 H COSY spectrum ( Figure 7 ), δ H 6.71 was correlated with δ H 6.98 and δ H 7.36, δ H 6.71 was correlated with δ H 7.03 andδ H Related to 7.20, it is speculated that both benzene ring structures are tris-ortho-substituted. In addition, 1 H- 1 In the H- δ H H COSY spectrum, two spin-coupling fragments, -CH2-CH2-CH2- and CH3-CH-CH-, were also determined. Further, the fragments of o-dihydroxybenzoyl, propanediamine, and threonine were connected through HMBC experiments. In addition, characteristic methylene signals appeared at δ H 5.64 and Figure 8 ). δ H 5.64 and δ H 5.55 were correlated with δ C 78.0 in the HMQC spectrum ( 3 ), suggesting that this methylene is a sp δ H 5.64 and δ H 5.55 showed correlation signals with C-7 and C-2, and H-6 also showed correlation signals with C-7 and C-2. Therefore, it is speculated that a methylenedioxy group is attached to positions C-7 and C-2 ( Figure 9 ). Finally, through biogenetic inference, the threonine in this compound should be of the L-type. Finally, the structure of the compound was identified as 2,3-dihydroxy-N-(3-((2 R ,3 R )-3-hydroxy-2-(((Z)-8-hydroxy-4H-benzo[d][1,3]dioxin-4-ylidene)amino)butanamido)propyl)benzamide. Searching in the Scifinder database revealed it as a new compound, named serratiochelin E. The structural formula of serratiochelin E is as follows: .
[0034] Table 1. 1 H NMR and 13 C NMR data of serratiochelin E (DMSO- d 6, 500, 125 MHz, δ in ppm, J in Hz) Note: The signals of active hydrogens (OH, NH) are not summarized in Table 1.
[0035] Evaluation of the anti-inflammatory activity of serratiochelin E in Example 4 RAW 264.7 mouse mononuclear macrophages with normal morphology and good condition were selected for activity evaluation. First, the cytotoxicity of serratiochelin E was determined by the CCK-8 method, and normal group, LPS group and experimental groups with different concentrations were set up. The results are as Figure 10 shown. At the detected concentrations (0.1 - 100 μ μM, prepared with DMSO), serratiochelin E did not show obvious cytotoxicity. A suspension with a cell density of about 5×10 5 cells / ml was prepared with DMEM medium and inoculated into a 96-well cell culture plate. 200 μ μL of the cell suspension was added to each well for plating, and 200 μ μL / well of 1×PBS (pH 7.2) was added around it. It was placed in a cell culture incubator with 5% CO2 at 37 °C for the cells to adhere and grow. When the cell state was stable and completely adhered, the supernatant in the wells was carefully discarded with a pipette. 100 μ μL of DMEM basal medium was added to the normal group and the LPS group, and 100 μ μL of DMSO solutions of serratiochelin E with different concentrations were added to each experimental group. After continued culture for 4 h, the supernatant in the wells was discarded. 200 μ μL of DMEM basal medium was added to the normal group, 200 μ μL of LPS solution with a final concentration of 300 ng / mL was added to the LPS group, and 100 μ μL of serratiochelin E solutions with different concentrations and 100 μ μL of LPS solution with a final concentration of 300 ng / mL were added to each experimental group. After the treatment, it was placed in a cell culture incubator with 5% CO2 at 37 °C for 24 h. After 24 h, the cell culture supernatant was taken for detection. The detection results are as Figure 11 shown. Serratiochelin E has the best anti-inflammatory effect at 50 μM - 100 μM and has a certain anti-inflammatory effect at 100 nM - 10 μM.
[0036] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.
Claims
1. A Serratia marcescens F2-2, characterized in that: Its classification name is Serratia. Marcescens , and it was deposited in the Guangdong Microbial Culture Collection Center on February 6, 2025, with the deposit number GDMCC No. 65860.
2. Use of Serratia marcescens F2-2 as claimed in claim 1 in the fermentative preparation of the siderophore compound Serratiochelin E, characterized in that: The structural formula of the said Serratiochelin E is as follows: 。 3. A method for preparing the siderophore compound Serratiochelin E, characterized in that: It includes the following steps: (1) Inoculate the activated Serratia marcescens F2-2 into SDA liquid medium and culture to obtain a seed fermentation broth; (2) Inoculate the seed fermentation broth obtained in step (1) into rice medium and statically culture at room temperature for 4 weeks to obtain F2-2 rice ferment; (3) Add an extraction solvent to the F2-2 rice ferment obtained in step (2), soak overnight and mash, obtain an extract by the two-step ultrasonic soaking extraction method, and obtain a dark red extract through reduced pressure concentration; (4) Dissolve the extract obtained in step (3), load it onto a normal-phase solid-phase extraction column, collect the fractions eluted with petroleum ether-acetone, load it onto a reverse-phase solid-phase extraction column, elute with a methanol-water system, and the eluate is separated by semi-preparative liquid phase after reduced pressure concentration to obtain Serratiochelin E; The structural formula of the said Serratiochelin E is: 。 4. The method for preparing the siderophore compound Serratiochelin E according to claim 3, characterized in that: The conditions of the culture in step (1) are to culture in a constant temperature shaking incubator at 28 °C and 150 r / min for 7 days.
5. The method for preparing the siderophore compound Serratiochelin E according to claim 3, characterized in that: The preparation method of the rice medium in step (1) is to soak 180 g of rice in 240 mL of distilled water overnight and sterilize at 121 °C for 20 min.
6. The method for preparing the siderophore compound Serratiochelin E according to claim 3, characterized in that: The extraction solvent in step (3) is ethyl acetate-methanol, and the ratio of material to liquid is 1:6; the extraction time is 30 min.
7. The method for preparing the siderophore Serratiochelin E according to claim 6, characterized in that: The volume ratio of the said ethyl acetate-methanol is 85:
15.
8. The method for preparing the siderophore compound Serratiochelin E according to claim 3, characterized in that: The conditions for the semi-preparative liquid separation described in step (4) are as follows: the chromatographic column is Reprosil Gold 120 C 18 , the mobile phase is 55% methanol-water isocratic elution, the flow rate is 1.5 mL / min, the injection volume is 100 μL, the detection wavelength is 210 nm, and the retention time is 45 min.
9. Use of Serratiochelin E obtained by the preparation method according to any one of claims 3-8 in the preparation of anti-inflammatory drugs.