Method for extracting and separating daucosterol from marine streptomyces L120
Through the method of extracting and isolating carotene from Streptocytica L120, the problem of difficulty in effectively extracting carotene from microorganisms in the prior art was solved, efficient and pure carotene separation was achieved, and the foundation for increasing yield in the later stage was laid.
Patent Information
- Application Number
- CN202510324774.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-19
- Publication Date
- 2025-06-20
AI Technical Summary
The prior art is difficult to effectively extract and isolate carotene from secondary metabolites of microorganisms, and the yield is low.
Pure carrosides were obtained by extracting and separating carrosides from Streptocytica L120, including strain activation and preservation, seed liquid culture, fermentation culture and extraction separation, and separation of secondary metabolites.
The efficient separation of pure carotene from Streptocytica L120 was achieved. The separation process was simple, the extraction and elution solvent used was low toxicity, and the obtained carotene was high in purity, few impurities, easy to refine, and high yield.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of preparation of daucosterol, and particularly to a method for extracting and separating daucosterol from marine streptomyces L120. Background Art
[0002] Daucosterol is a phytosterol glycoside widely present in plants. After separation, it has been successively used in the research of pharmacological effects and disease prevention and treatment. Research shows that daucosterol can effectively limit the migration and invasion of human liver cancer cells and human breast cancer cells within a certain concentration range, indicating that daucosterol has a certain anti-tumor effect. At the same time, daucosterol can protect human microvascular endothelial cells from oxidative damage. After comparing the repair effects on oxidized damaged cells, it is found that daucosterol shows a repair and treatment effect, but the protection is better than the repair effect, that is, prevention is better than treatment. Daucosterol belongs to natural steroid compounds. The research and preparation of daucosterol play an important role and have broad medical prospects for the development of natural active steroid compound drugs. Daucosterol components are contained in many plants in nature, but the content is relatively low, usually obtained during the systematic separation of plant components. At present, there are few reports on the separation of daucosterol from the secondary metabolites of microorganisms, and the yield is generally low.
[0003] Genistein, also known as genistein, as a protein kinase inhibitor, can prevent pathogenic bacteria from invading mammalian epithelial cells and has a certain inhibitory effect on pathogenic bacteria. At the same time, genistein also has a certain effect on inhibiting tumors, and genistein can inhibit the growth of a variety of tumor cells and induce apoptosis of a variety of tumor cell lines. Genistein inhibits the growth of tumor cells by regulating genes related to cell cycle and apoptosis homeostasis, and can inhibit the nuclear transcription factor NF-κB and Akt signaling pathways that maintain the balance of cell survival and programmed cell death (apoptosis). In addition, genistein is also an effective inhibitor of angiogenesis and metastasis. Genistein has made remarkable progress in both in vivo and in vitro studies and is a promising chemical substance for preventing or treating tumors. However, so far, most of the sources of genistein mainly come from plants, and there are few found from microorganisms. Summary of the Invention
[0004] In order to solve the technical problem of difficult extraction and separation of daucosterol from the secondary metabolites of microorganisms, the present invention discloses a method for extracting and separating daucosterol from marine streptomyces L120.
[0005] To achieve the above object, the present invention adopts the following technical solutions:
[0006] Step 1, Strain Activation and Preservation
[0007] Take Streptomyces maritimus L120 out of the -80°C refrigerator. Take 100 μL of the bacterial liquid and spread it on a solid medium. Then place the petri dish in an incubator at a constant temperature of 28°C for 3 days to grow spores.
[0008] After the seed liquid culture is completed, use a pipette to suck 1 mL of the seed liquid in a laminar flow hood and place it in a sterile cryopreservation tube. Then add 1 mL of sterilized 40% glycerol, mix well, and store it in the -80°C refrigerator for later use.
[0009] Step 2, Seed liquid culture
[0010] In a laminar flow hood, use a sterilized pipette tip or inoculation loop to select a bacterial block with a size of 1 / 8 of the solid plate and inoculate it into a TSB liquid medium to obtain a seed liquid. Then take 100 mL of the seed liquid and put it into a 1000 mL conical flask with baffles, and culture it on a shaker for 16 h.
[0011] Step 3, Fermentation culture and extraction separation of the strain
[0012] 3.1 Transfer the well-grown seed liquid to the M33 fermentation medium at an inoculation amount of 10%, and use a fermenter for on-tank fermentation for 5 days.
[0013] 3.2 After fermentation is completed, centrifuge at a rotational speed of 8000 rpm and a temperature of 4°C to obtain two parts: bacterial liquid and mycelium; add 3 times the volume of methanol to the mycelium and extract it 3 times. Evaporate and concentrate the mycelium extract to obtain a total of 55 g of methanol crude extract paste; Step 4, Separation of secondary metabolites
[0014] Take the methanol crude extract paste, add a small amount of methanol and dissolve it by ultrasonic treatment, then stir it well with G200 - 300 mesh silica gel, and vacuum dry to remove the solvent; pack the silica gel with the crude product into the upper layer of a G300 - 400 mesh silica gel column, and then use a dichloromethane - methanol system as the mobile phase for gradient elution, collect the eluate in fractions, separate to obtain a white solid, remove the upper liquid, and obtain the lower white precipitate part.
[0015] Then repeatedly rinse the white precipitate part with methanol to remove impurities to obtain pure daucosterol.
[0016] Furthermore, in step 1, the solid medium is selected from one of ISP2, ISP4, A1, Gao's No. 1 and TSB media.
[0017] Furthermore, in step 2, during the process of culturing the seed liquid on the shaker, the controlled conditions are: temperature is 28°C, rotational speed is 220 rpm, and humidity is 40% - 50%.
[0018] Further, in step 3.1, the fermentation culture conditions are as follows: the rotation speed is 399 rpm / min, the oxygen supply is 1%, and the temperature is 28°C; the components of the M33 medium (g / L) are: soluble starch 30, soybean powder 10, yeast extract 2.5, calcium carbonate 3, and pH 7.2 - 7.5.
[0019] Further, in step 4, the silica gel used is 300 - 400 mesh, and the size of the chromatographic column selected is 15 * 70 cm.
[0020] Further, in step 4, the volume ratios of dichloromethane - methanol are respectively: 1:0, 95:5, 10:1, 5:1, 3:1, 1:1, and 0:1.
[0021] The present invention also discloses a method for extracting and separating genistein from marine Streptomyces L120, and the separation method is as follows:
[0022] Step 1, strain activation and preservation
[0023] Take marine Streptomyces L120 out of the -80°C refrigerator, take 100 μL of the bacterial solution and spread it on the solid medium, then place the culture dish in a constant temperature incubator and culture for 3 days at a temperature of 28°C to grow spores;
[0024] After the seed liquid culture is completed, use a pipette to suck 1 mL of the seed liquid in a laminar flow hood, place it in a sterile cryopreservation tube, then add 1 mL of sterilized 40% glycerol, mix well and place it in the -80°C refrigerator for cryopreservation for later use;
[0025] Step 2, seed liquid culture
[0026] In a laminar flow hood, use a sterilized pipette tip or inoculation loop to select a bacterial block with a size of 1 / 8 of the solid plate, inoculate it into the TSB liquid medium to obtain the seed liquid, then take 100 mL of the seed liquid and put it into a 1000 mL conical flask with a baffle, and culture it on a shaker;
[0027] Step 3, fermentation culture and extraction separation of the strain
[0028] 3.1. Transfer the well - grown seed liquid to the M33 fermentation medium according to an inoculation amount of 10%, and use the method of fermenting in a fermenter for 5 days;
[0029] 3.2. After fermentation, centrifuge at a rotation speed of 8000 rpm and a temperature of 4°C to obtain two parts: the bacterial liquid and the mycelium; add 3 times the volume of methanol to the mycelium and extract it 3 times, evaporate and concentrate the mycelium extract to obtain a total of 55 g of methanol crude extract paste;
[0030] Step 4, separation of secondary metabolites
[0031] The crude methanol extract was added with a small amount of methanol and dissolved by ultrasonic treatment. Then it was thoroughly stirred and mixed with silica gel of 200 - 300 mesh, and the solvent was removed by vacuum drying. The silica gel with the crude product was packed into the upper layer of a silica gel column of 300 - 400 mesh, and then gradient elution was carried out using a dichloromethane - methanol system as the mobile phase. According to the different polarities of the samples on thin - layer chromatography, a total of ten components were obtained.
[0032] The fifth component separated by silica gel was further separated. The sample was separated by the molecular sieve effect and then passed through a silica gel column again. When the volume ratio of petroleum ether to ethyl acetate was 1:1, 15 mg of genistein was separated.
[0033] The beneficial effects of the present invention are as follows: Most of the daucosterol is obtained from plants, and it is rarely isolated from the secondary metabolites of microorganisms with low yields. This method separates pure daucosterol from the secondary metabolites of marine Streptomyces L120. The separation process is simple, without the need for repeated column chromatography separation. The process for preparing daucosterol is simple, the extraction and elution solvents used have low toxicity, the obtained daucosterol has high purity, few impurities, is easy to refine, and has a high yield. At the same time, it lays a foundation for improving the yield of daucosterol at the molecular biology level in the later stage.
[0034] The present invention also separates genistein from the secondary metabolites of marine Streptomyces L120, thus realizing the separation and extraction from microorganisms, providing a reference for subsequent research. Brief Description of the Drawings
[0035] Figure 1 It is a schematic diagram of the separation and purification experiment of daucosterol in the embodiment of the present invention;
[0036] Figure 2 It is the 1H - NMR spectrum of daucosterol in the embodiment of the present invention
[0037] Figure 3 It is the 13C - NMR spectrum of daucosterol in the embodiment of the present invention;
[0038] Figure 4 It is the low - resolution mass spectrum of daucosterol in the embodiment of the present invention;
[0039] Figure 5 It is the phylogenetic tree of strain L120 in the embodiment of the present invention;
[0040] Figure 6 It is the genomic circular map of strain L120 in the embodiment of the present invention;
[0041] Figure 7 It is the HPLC chromatogram of genistein in the embodiment of the present invention;
[0042] Figure 8 It is the 1H - NMR spectrum of genistein in the embodiment of the present invention;
[0043] Figure 9 This is the carbon-13 NMR spectrum of genistein in the embodiments of the present invention. Detailed implementation manners
[0044] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0045] There are few reports on the isolation of daucosterol from Streptomyces. The vast majority is obtained from plants, and there are very few other disclosed methods. The present invention starts from Streptomyces L120, which has a genome capable of producing daucosterol. It is found that daucosterol can be produced, isolated and obtained from the fermentation of Streptomyces L120.
[0046] 1. Genome sequencing, assembly and analysis of Streptomyces L120
[0047] Through whole-genome sequencing, it is determined by analysis that the L120 genome consists of 6 different sequences, including five plasmids with linear structures. This result not only expands the understanding of the genetic diversity of this strain, but also reveals its unique gene vector characteristics. The GC content of the L120 genome is 71.84%, which is consistent with the corresponding values of other members of the Streptomycetaceae family, reflecting its adaptation to genetic stability in evolution. A high GC content is usually closely related to the environmental adaptability, metabolic mechanisms and gene regulation methods of bacteria, further suggesting that L120 may have evolved unique metabolic pathways and survival mechanisms in its specific ecological niche.
[0048] The present invention also details the sequence details including sequence length, identification codes and their potential functions, etc., aiming to provide a solid foundation for subsequent structure and function research. The details of these sequences provide key information for further exploring the metabolic potential of L120 and its applications in the field of biotechnology in the future. Through the comprehensive analysis of these sequences, we hope to analyze the adaptation strategies of L120 in the natural environment and the potentially beneficial bioactive molecules it may produce, which has potential value for the development of new antibiotics and other bioactive compounds.
[0049] Table 1 General situation of the genome of strain L120
[0050]
[0051] Table 2 Genome distribution of strain L120
[0052]
[0053] 2. Strain systematic classification and identification
[0054] The phylogenetic NJ tree based on 16S rRNA sequences showed that Streptomyces L120 was closely related to Streptomyces bacillaris. As Figure 5 shown, and it formed a separate branch. This indicates that the 16S rRNA identity of Streptomyces L20 with these species is relatively low, and it is very likely to be a new strain, which still needs further verification.
[0055] The OrthoANI algorithm was used to perform pairwise genomic computational analysis on the genomes of 10 closely related Streptomyces species, aiming to deeply explore and compare the genetic similarities among these species. By analyzing each genomic sequence in detail and using the OrthoANI value as a measure, the genetic relationships among these Streptomyces species were inferred. The analysis results were clearly shown in the corresponding graphical representation, which specifically pointed out that the OrthoANI value between sample L120 and Streptomyces bacillaris was significantly higher than that of other comparison groups. This finding once again emphasizes the high genetic similarity between L120 and Streptomyces bacillaris, thus supporting the conclusion that L120 belongs to Streptomyces bacillaris.
[0056] 3. Biosynthetic gene cluster analysis
[0057] Circular genome map of Streptomyces bacillaris L120 Figure 6From the inside out, the name of the strain is in the exact middle of the circular genome. The first circle represents the scale; the second circle represents the GC Content; the third circle represents the GC Skew; the fourth circle represents the positions of CDS, tRNA, rRNA, biosynthetic gene clusters, and the compound types of various secondary metabolites. It can be seen that the secondary metabolites of this strain are relatively rich. The antiSMASH 6.0 was used to predict the biosynthetic gene clusters of secondary metabolites in the genome of strain L120. The analysis of the secondary metabolites of the strain by antiSMASH found that the strain included at least 32 secondary metabolite synthesis gene clusters, and the secondary metabolites were rich, including 3 NRPSs, 8 PKSs, 1 NRPS-PKS hybrid, 1 Saccharide-PKS hybrid, and 3 terpenes. It can be seen that strain L120 contains more NRPS compounds. The NRPS-type BGCs in the strain were compared and analyzed using the BLAST tool of NCBI to estimate the functional roles of the core genes, and further determine the amino acid assembly through bioinformatics software such as PRISM, NaPDoS, and PKS / NRPS, and conduct a preliminary exploration of NRPS compounds at the molecular level.
[0058] 4. Genome Prediction
[0059] The exploration of bacterial gene islands is crucial for revealing the mechanisms of microbial genetic diversity, evolutionary dynamics, pathogenic mechanisms, and the spread of drug resistance. These DNA fragments are rich in gene clusters related to specific biological functions, such as pathogenicity, antibiotic resistance, etc., providing bacteria with means to adapt to environmental pressures and enhance their survival competitiveness. In-depth study of gene islands not only helps to explain pathogen mechanisms and develop new treatment plans in the medical field, but also has important significance in the fields of microbiology and evolutionary biology for understanding horizontal gene transfer (HGT) events and the formation of genetic diversity among microorganisms.
[0060] Table 5 Gene Island Distribution of Strain L120
[0061]
[0062] In molecular biology research, the identification and analysis of promoter regions are key steps in understanding the gene expression regulation mechanism. The change in the average free energy of the promoter region is closely related to the GC content of the flanking regions. Based on this, we used the PromPredict v1 tool and set a series of free energy-based thresholds to comprehensively analyze the microbial genomic DNA with different GC contents. This strategy aims to establish a set of universal criteria for efficiently predicting promoter regions in microbial genomes. Further prediction work focused on the sequences within 500 bp upstream of the genes and screened out promoters with a prediction confidence level higher than level2, aiming to improve the accuracy and reliability of the prediction. This method not only covers the sense and antisense strands of the genome but also provides important data support for in-depth research on microbial gene expression regulation. The results are shown in the following table, providing a solid foundation for subsequent research.
[0063] Table 6 Promoter prediction of strain L120
[0064]
[0065] Example
[0066] The present invention discloses a method for extracting and isolating daucosterol from marine Streptomyces L120, which specifically includes the following steps:
[0067] 1 Strain activation and preservation
[0068] Take out marine Streptomyces L120 from the -80°C refrigerator, take 100 μL of the bacterial solution and spread it on a solid medium, then place the culture dish in a constant temperature incubator and culture for 3 days at a temperature of 28°C to grow spores;
[0069] After the seed liquid culture is completed, use a pipette to suck 1 mL of the seed liquid in a biosafety cabinet, place it in a sterile cryotube, and then add 1 mL of sterilized 40% glycerol. Mix well and store it in the -80°C refrigerator for later use.
[0070] The solid medium is selected from one of ISP2, ISP4, A1, Gao's No. 1, and TSB media.
[0071] 2 Seed liquid culture
[0072] In a biosafety cabinet, use a sterilized pipette tip or inoculation loop to select a bacterial block with a size of 1 / 8 of the solid plate and inoculate it into the TSB liquid medium to obtain the seed liquid. Then take 100 mL of the seed liquid and put it into a 1000 mL conical flask with baffles and culture it on a shaker.
[0073] During the culture of the seed liquid on the shaker, the control conditions are: temperature is 28°C, rotation speed is 220 rpm, and humidity is 40% - 50%.
[0074] Fermentation Culture and Extraction and Separation of Strain 3
[0075] 3.1 Transfer the well-grown seed liquid to the M33 medium according to an inoculation amount of 10%, and carry out fermentation in a fermenter for 5 days by the method of upper-tank fermentation.
[0076] Composition of M33 medium: soluble starch 30, soybean powder 10, yeast extract 2.5, calcium carbonate 3, pH 7.2 - 7.5.
[0077] 3.2 After fermentation is completed, centrifuge at a rotation speed of 8000 rpm and a temperature of 4°C to obtain two parts: the bacterial liquid and the mycelium; add 3 times the volume of methanol to the mycelium and extract 3 times, evaporate and concentrate the mycelium extract to obtain a total of 55 g of methanol crude extract paste.
[0078] Fermentation culture conditions: rotation speed is 399 rpm / min, oxygen supply is 1%, and temperature is 28°C.
[0079] 4. Separation of Secondary Metabolites
[0080] Soak 2000 g of silica gel thoroughly with dichloromethane, load it into a chromatographic column, tap the surroundings with an ear bulb during the packing process, and use the dichloromethane - methanol system as the eluent for gradient elution. After separation, a white solid is obtained. Remove the upper liquid to obtain the lower white precipitate part.
[0081] Then repeatedly rinse the white precipitate part with methanol to remove impurities, and pure daucosterol can be obtained.
[0082] The silica gel selected is 300 - 400 mesh, and the size of the chromatographic column selected is 15 * 70 cm.
[0083] Among them, in the elution system, the volume ratio of dichloromethane to methanol is shown in Table 7 below.
[0084] Table 7
[0085]
[0086]
[0087] When the volume ratio of dichloromethane:methanol is 10:1, the eluent is evaporated and concentrated to obtain an elution fraction containing daucosterol, as Figure 1 shown. The compound is determined to be daucosterol by nuclear magnetic resonance and its mass spectrum, as Figures 2 - 4 shown.
[0088] Example 2
[0089] In addition, 15 mg of genistein was also obtained from the fermentation product of the marine Streptomyces bacillaris L120.
[0090] The separation method is as follows: 2000 g of silica gel was fully soaked in dichloromethane and loaded into a chromatography column. During the packing process, an ear bulb was used to tap around. A dichloromethane-methanol system was used as the eluent for gradient elution. According to the different polarities of the sample thin-layer chromatography, it was divided into ten components in total.
[0091] At first, the fifth component separated by silica gel was further separated. First, an LH20 gel column was used, as shown in Table 8. The sample was separated using the molecular sieve effect, and then through a silica gel column. When the volume ratio of petroleum ether to ethyl acetate was 1:1, genistein was separated out.
[0092] Under the ultraviolet wavelength of 280 nm, the high-performance liquid chromatography of genistein, after nuclear magnetic resonance determination, as Figures 7 - 9 shown, it was determined that this substance was genistein.
[0093] Table 8 L120 Liquid Phase Program
[0094] Time (min) Phase A (Chromatographic Methanol) Phase B (Ultra-pure Water) 0 min 15％ 85％ 40 min 100％ 0％ 42 min 100％ 0％ 44 min 15％ 85％ 45 min 15％ 85％
[0095] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the essence of the present invention should also fall within the protection scope of the present invention.
Claims
1. A method for extracting and isolating carotene from marine Streptomyces L120, characterized in that: The specific steps include: Step 1, strain activation and preservation Take out marine Streptomyces L120 from the -80°C freezer, take 100 μL of the bacterial solution and spread it on the solid culture medium, then place the culture dish in a constant temperature incubator for 3 days at 28°C to grow spores; After the seed solution culture is completed, use a pipette to aspirate 1 mL of seed solution in a clean bench, place it in a sterile cryopreservation tube, add 1 mL of sterilized 40% glycerol, mix well, and place in a -80°C refrigerator for later use; Step 2: Seed liquid culture In a clean bench, use a sterilized pipette tip or inoculation loop to select a bacterial block 1 / 8 the size of a solid plate, inoculate it into TSB liquid culture medium to obtain a seed solution, then take 100 mL of the seed solution and put it into a 1000 mL conical flask with a baffle, and culture it on a shaker for 16 hours; Step 3: Fermentation and extraction of strains 3.
1. Transfer the grown seed liquid to M33 fermentation medium at a 10% inoculation rate, and ferment for 5 days using a fermentation tank. 3.
2. After fermentation, centrifuge at 8000 rpm and 4°C to obtain a bacterial solution and mycelium; add 3 times the volume of methanol to the mycelium for extraction 3 times, evaporate and concentrate the mycelium extract to obtain a total of 55 g of methanol crude extract; Step 4. Separation of secondary metabolites Take a crude methanol extract and add a small amount of methanol to dissolve it by ultrasonication, then stir and mix it with G200-300 mesh silica gel, and vacuum dry it to remove the solvent; and fill the silica gel with the crude product into the upper layer of G300-400 mesh silica gel column, and then use dichloromethane-methanol system as mobile phase for gradient elution, collect the eluent in sections, and obtain a white solid after separation, remove the upper liquid, and obtain the lower white precipitate part; The white precipitate is then repeatedly washed with methanol to remove impurities and obtain pure carotene.
2. The method for extracting and isolating carotene from marine Streptomyces L120 according to claim 1, characterized in that: In step 1, the solid culture medium is selected from one of ISP2, ISP4, A1, Gao's No. 1 and TSB medium.
3. A method for extracting and isolating carotene from marine Streptomyces L120 as claimed in claim 2, characterized in that: In step 2, during the cultivation of the seed solution on the shaking table, the control conditions are: temperature of 28° C., rotation speed of 220 rpm, and humidity of 40%-50%.
4. A method for extracting and isolating carotene from marine Streptomyces L120 as claimed in claim 3, characterized in that: In step 3.1, the fermentation culture conditions are: rotation speed of 399 rpm / min, oxygen flow rate of 1%, and temperature of 28°C; M33 culture medium ingredients g / L: soluble starch 30, soybean powder 10, yeast extract 2.5, calcium carbonate 3, pH 7.2-7.
5.
5. A method for extracting and separating carotene from marine Streptomyces L120 as claimed in claim 4, characterized in that: In step 4, the selected silica gel is 300-400 mesh, and the selected chromatographic column size is 15*70cm.
6. A method for extracting and isolating carotene from marine Streptomyces L120 as claimed in claim 5, characterized in that: In step 4, the volume ratios of dichloromethane to methanol are: 1:0, 95:5, 10:1, 5:1, 3:1, 1:1, 0:1, respectively.