Method for directly preparing high-purity and high-activity prodigiosin from fermentation liquor

Abstract

The invention belongs to the field of prodigiosin extraction, and discloses a method for directly preparing high-purity and high-activity prodigiosin from a fermentation broth, which comprises the following steps: mixing thallus precipitate separated from the fermentation broth with an acidic alcohol solvent, carrying out ultrasonic treatment, leaching and separation, and concentrating the obtained supernatant to obtain a prodigiosin crude product; performing semi-preparative liquid chromatography separation after filtration and sample loading, performing elution by using a mobile phase, monitoring a chromatographic peak in real time, and collecting fractions of target prodigiosin according to detection signal intensity and / or effluent color change; concentrating and centrifuging to remove supernatant to obtain prodigiosin precipitate, and drying to obtain powdery prodigiosin. According to the method, the extremely simple process of directly preparing the high-purity and high-activity prodigiosin from the fermentation liquor is completed within 1.5 h or less, the prodigiosin with the purity larger than or equal to 99% is directly obtained from the fermentation liquor, the pharmacopoeia-grade purity can be achieved through single equipment, single solvent and single operation, operation is easy and convenient, conditions are stable, and implementation is easy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of prodigiosin extraction, and particularly relates to a method for efficiently separating and purifying prodigiosin from prodigiosin fermentation liquor. BACKGROUND

[0002] Prodigiosin (PG) is a natural red tripyrrole secondary metabolite produced by a variety of microorganisms (such as Serratia marcescens). As a natural pigment, prodigiosin has potential application value in the field of textile printing and dyeing. More importantly, prodigiosin exhibits a variety of significant biological activities, especially in antibacterial, antitumor and other aspects, such as inducing colorectal cancer cell apoptosis, and thus is considered to have the potential to develop new anticancer drugs and other applications in the pharmaceutical field. In addition, its unique pH-responsive color change characteristics also make it have broad application prospects in the field of material science, such as intelligent labeling, packaging materials and special substrates.

[0003] In recent years, with the increasing preference of global consumers for healthy and natural products, and the continuous exploration of the application potential in the fields of biological medicine and high-end materials, the market demand for prodigiosin has shown a rapid growth trend. At present, the industrial scale production of prodigiosin mainly adopts the microbial fermentation method. In this method, Serratia marcescens is commonly used as the production strain. However, the composition of the fermentation liquor obtained after the fermentation process is extremely complex, the concentration of the target product prodigiosin is usually low, and it also contains a large amount of cell debris, proteins, polysaccharides and other metabolic by-products and impurities. This complex matrix brings great challenges to the subsequent separation and purification of prodigiosin.

[0004] In the existing purification method of prodigiosin, silica gel column chromatography is a commonly used technical means. However, this method has many shortcomings in practical application: first, the purity problem is prominent. The traditional silica gel column chromatography method often cannot effectively remove the complex impurities in the fermentation broth, resulting in low purity of the obtained prodigiosin product, usually difficult to reach 90%, which greatly limits its application in high value-added fields, especially for medical use which requires extremely high purity. Second, the yield loss is serious. In the separation and purification process, prodigiosin may not only cause physical loss due to insufficient adsorption and elution, but more importantly, the pigment is sensitive to light, oxygen and certain chemical conditions, and is prone to oxidative degradation and other chemical losses, resulting in low actual yield of the final product, usually less than 85%, or even lower, which directly affects the economic benefit of production. Third, the time and economic cost is high. The traditional silica gel column chromatography method usually needs a long time for equilibration, sample loading, elution and collection, and the whole purification cycle is long, which seriously restricts the production efficiency. At the same time, a large amount of organic solvent may be used in this method, increasing the production cost and causing certain burden to the environment, which does not meet the current requirements of green chemistry and sustainable development.

[0005] In summary, it is a key bottleneck technical problem that restricts the further development and growth of prodigiosin industry to efficiently extract and purify prodigiosin from fermentation broth with high purity, high yield, while taking into account the greenness, economy (including time cost and material cost), simplicity of operation, stability and repeatability of the process. The existing technology cannot meet the multiple demands of high purity, high yield, low cost, short cycle, green environmental protection, good stability and simple operation at the same time, therefore, it is urgent to develop a new type of prodigiosin separation and purification technology with better comprehensive performance to meet the growing market demand and promote the industrialization process. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the above background art, and to provide a simple method for directly preparing high-purity, high-activity prodigiosin from fermentation broth, which can effectively obtain high-purity prodigiosin.

[0007] To solve the above technical problems, the technical solution provided by the present application is: A method for directly preparing high-purity, high-activity prodigiosin from fermentation broth, comprising the following steps: (1) performing solid-liquid separation on the fermentation broth containing prodigiosin to obtain a cell precipitate, mixing the cell precipitate with an acidic alcohol solvent, performing ultrasonic treatment, leaching and solid-liquid separation to obtain a supernatant containing prodigiosin, and concentrating to obtain a prodigiosin crude product; (2) The crude styraxin was filtered to obtain a sample solution, which was then loaded and separated by semi-preparative liquid chromatography. The mobile phase was used for elution, the chromatographic peaks were monitored in real time, and the fraction of the target styraxin was collected according to the intensity of the detection signal and / or the color change of the effluent. (3) The collected styraxin fraction was concentrated, the supernatant was removed by centrifugation, and styraxin precipitate was obtained. After drying, styraxin powder was obtained.

[0008] The above method, further, in step (1), the fermentation broth containing strychnine is Serratia marcescens (… Serratia marcescens Fermentation broth.

[0009] Furthermore, the *Serratia marcescens* ( Serratia marcescens The preparation method of fermentation broth includes the following steps: inoculating plate-activated Serratia marcescens into seed culture medium, and obtaining the inoculated strain after the strain grows to the logarithmic growth phase; inoculating the inoculated strain into antibiotic-free LB liquid culture medium, and shaking culture in a shaker at a temperature of 27~31℃ (preferably 29℃) and a rotation speed of 150~170 rpm for 24~48h to obtain Serratia marcescens fermentation broth.

[0010] Furthermore, in step (1), the acidic alcohol solvent is chromatographic grade methanol with a pH value of 3.0~4.0, and the mass ratio of the bacterial precipitate to the acidic alcohol solvent is 1:8~12. Furthermore, in step (1), the ultrasonic treatment power is 250W~350W, and the ultrasonic time is 8min~12min; the extraction is carried out at room temperature for 1.5h~2.5h; the centrifugation speed for solid-liquid separation is 7000rpm~9000rpm, and the centrifugation time is 8min~12min; the ultrasonic treatment, extraction, and solid-liquid separation steps are repeated until the color of the bacterial precipitate turns purplish-gray, and all supernatants are combined.

[0011] Furthermore, in step (1), the concentration is carried out by rotary evaporation, and the rotary evaporation temperature is 40~45℃.

[0012] Furthermore, in step (2), the filter membrane used for filtration is a filter membrane with a pore size ≤ 0.45 μm (such as a 0.22 μm or 0.45 μm filter membrane) to obtain a sample solution suitable for loading into a semi-preparative liquid chromatography system.

[0013] Further, in step (2), the semi-preparative liquid chromatography separation conditions include: using a C18 chromatographic column and an ultraviolet detector, setting the detection wavelength to 535 nm, the flow rate is 4.5-5.0 mL / min, the sample loading amount is 1-5 mL, using isocratic elution mode, and the eluent is a mixture of water containing 0.1% trifluoroacetic acid by volume and chromatographic grade methanol at a volume ratio of 25-35:65-75. The semi-preparative liquid chromatography system supports full automatic / manual dual mode, and the key parameters can be set by one key to reproduce, significantly reducing manual intervention and on-duty time, improving batch turnover efficiency, and reducing labor intensity.

[0014] Further, in step (2), according to the detection signal intensity and / or the color change of the effluent, specifically: when the intensity of the chromatographic peak is 50-200 mAU, the retention time is 20-50 min, and / or when the effluent color starts to be red, the high-purity fraction of the prodigiosin target is collected.

[0015] Further, in step (2), the concentration is performed by rotary evaporation concentration, and the rotary evaporation concentration temperature is 30-35℃.

[0016] Compared with the prior art, the beneficial effects of the present application are: 1. The present application realizes a simple process for directly preparing high-purity and high-activity prodigiosin from fermentation broth within ≤1.5 h by using the optimized semi-preparative liquid chromatography method, and the purity of the prodigiosin obtained directly from the fermentation broth is ≥99%, which is more than 50% shorter than the traditional silica gel column chromatography-multiple solvent switching route, and the present application realizes the pharmacopoeia grade purity by using a single device, a single solvent and a single operation, which is simple to operate, stable in conditions and easy to realize.

[0017] 2. The methanol-dilute trifluoroacetic acid system used in the present application meets the ICH Q3C three-class solvent requirements, there are only two kinds of organic solvents in the whole process, the total amount is reduced by about 70%, and there is no halogenated hydrocarbon, the flash point is high, the occupational exposure limit is loose, the liquid chromatography system is relatively closed, which further reduces the direct contact between the operator and the organic solvent and reduces the potential health risk; the online solvent recovery rate is >90%, the energy consumption and waste discharge are reduced synchronously, and the GMP amplification, low investment, low energy consumption and green economic demand are considered, which provides a stable, friendly and sustainable technical solution for the industrialized high-purity preparation of prodigiosin. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creating any inventive labor.

[0019] Figure 1 Preparation flow chart of the embodiment of the present application; Figure 2 Sample preparation liquid phase chart of the embodiment of the present application; Figure 3 Preparation liquid phase collection different stage fraction chart of the embodiment of the present application; Figure 4 Proflavine flocculent state solution of the embodiment of the present application; Figure 5 PG standard substance standard curve of the embodiment of the present application; Figure 6 Qualitative chromatogram of the self-extracted substance by the analytical liquid chromatography of the embodiment of the present application; Figure 7 Purity chart of the self-extracted proflavine by the thin layer chromatography of the embodiment of the present application; Figure 8 Purity chart of the self-extracted proflavine by the LC-MS of the embodiment of the present application; Figure 9 Purity of the self-extracted PG after purification of the present application; Figure 10 Purity and peak time effect chromatogram of the extracted substance by the analytical liquid chromatography under the optimized mobile phase condition of the embodiment of the present application; Figure 11 Proliferation inhibition effect of the self-extracted proflavine biological activity effect research of the embodiment of the present application; Figure 12 Promoting apoptosis effect of the self-extracted proflavine biological activity effect research of the embodiment of the present application; Figure 13 Inhibiting tumor progression effect of the self-extracted proflavine biological activity effect research of the embodiment of the present application; Figure 14 Preparation sample liquid chromatogram before and after purification of the embodiment of the present application. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present application is not limited to the following specific embodiments.

[0021] Unless otherwise defined, all the professional terms used herein have the same meaning as generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing the specific embodiments, and are not intended to limit the scope of protection of the present application.

[0022] Unless otherwise specifically explained, various materials, reagents, instruments and equipment that are used in the present application are commercially available or are prepared by known methods.

[0023] The present application provides a method for purifying high purity prodigiosin from a fermentation broth containing prodigiosin, which comprises the following steps: (1) Fermentation broth pretreatment and crude extraction: The fermentation broth containing prodigiosin is subjected to a first solid-liquid separation (e.g., by centrifugation or filtration) to obtain a cell pellet and a fermentation broth supernatant.

[0024] The cell pellet is mixed with an acidic alcoholic solvent (preferably chromatographic grade methanol), subjected to ultrasonic treatment to assist in cell wall disruption, followed by leaching, and then subjected to a second solid-liquid separation to obtain a supernatant containing prodigiosin. The ultrasonic treatment, leaching, and solid-liquid separation steps are repeated until the color of the cell pellet becomes purple-gray, and the supernatants obtained in each step are combined to form a first prodigiosin crude product. The first prodigiosin crude product (i.e., the combined supernatant) is subjected to rotary evaporation concentration and volume reduction to obtain a second prodigiosin crude product.

[0025] (2) Sample preparation: The obtained second prodigiosin crude product is filtered (e.g., using a 0.22 μm or 0.45 μm filter membrane) to remove insoluble impurities, obtaining a sample solution suitable for loading onto a semi-preparative liquid chromatography system.

[0026] Semi-preparative liquid chromatography purification: The filtered sample solution is injected into a semi-preparative liquid chromatography system. The system typically includes a manual injector, a detector (preferably a UV detector with a detection wavelength set at the maximum absorption wavelength of prodigiosin, such as 535 nm), a chromatography column (preferably a C18 reverse phase column, such as a 10 mm × 250 mm size), and a fraction collection area.

[0027] Isocratic elution is performed using a mobile phase at a flow rate of 4.5-5.0 mL / min, with a loading amount of 1-5 mL. The eluent is a mixture of 0.1% (by volume) trifluoroacetic acid in water and chromatographic grade methanol at a volume ratio of 25-35:65-75. Under these conditions, prodigiosin forms a major chromatographic peak on the chromatogram.

[0028] The key advantage of the method of the present application lies in the simplicity of the purification procedure and the ability to monitor in real time: during the chromatographic run, the operator can monitor the signal changes of the detector (intensity of the chromatographic peak 50~200 mAU and retention time 20~50 min) in real time, while the color change of the effluent (prodigiosin is red) can be observed visually. Combining these two pieces of information, the target prodigiosin fraction with high purity can be collected manually or automatically. This method of visual real-time collection based on chromatographic analysis significantly improves the accuracy and purity of the target fraction collection, avoiding the mixing of impurities, and embodies the high efficiency and precision of the purification method.

[0029] (3) Post-treatment and drying: Mix the collected high-purity prodigiosin fraction evenly, and perform vacuum concentration (for example, rotary evaporation) to remove most of the solvent (mainly methanol), obtaining a concentrated aqueous solution or semi-solid material containing prodigiosin.

[0030] Perform high-speed centrifugation (for example, 10000 rpm, 8 min) on the concentrated solution, separate and discard the supernatant, and obtain the prodigiosin precipitate, which helps to further remove residual water-soluble small molecular impurities.

[0031] Perform vacuum freeze-drying on the obtained prodigiosin precipitate to remove residual water, and finally obtain a high-purity prodigiosin product in powder form.

[0032] Regarding the preferred source of the starting fermentation broth: Preferably, the prodigiosin-containing fermentation broth is a fermentation broth of Serratia marcescens. Serratia marcescens The preparation method of the Serratia marcescens fermentation broth can include the following steps: inoculating plate-activated Serratia marcescens into a seed culture medium, allowing the strain to grow to the logarithmic growth phase to obtain an inoculated strain; inoculating the inoculated strain into an antibiotic-free LB (Luria-Bertani) liquid culture medium at a volume percentage of 0.5%, and culturing in a shaking incubator at a temperature of 29°C and a rotation speed of 160 rpm for 24~48 h to obtain the Serratia marcescens fermentation broth. During this process, acidic analytical methanol can be used for auxiliary extraction to improve the initial yield of prodigiosin. In order to protect prodigiosin from photodegradation, the steps of rotary evaporation concentration and the like are preferably carried out in the dark, for example, the temperature of the first vacuum distillation is controlled at 40~45°C, and the temperature of the second vacuum distillation is controlled at 30~35°C.

[0033] Example: A method for directly preparing high-purity, high-activity prodigiosin from a fermentation broth, the full flow chart is shown in Figure 1 , and the specific steps are as follows: 1. Preparation of fermentation broth: The fermentation broth was prepared by activating Serratia marcescens on agar plates (…). Serratia marcescens The inoculated strain was inoculated into the seed culture medium and allowed to grow to the logarithmic growth phase to obtain the inoculated strain. The inoculated strain was then inoculated into antibiotic-free LB liquid culture medium at a volume percentage of 0.5%. The culture was carried out at 29°C and 160 rpm for 24 hours to obtain the fermentation broth of Serratia marcescens.

[0034] 2. First solid-liquid separation: The above Serratia marcescens fermentation broth was centrifuged at 8000 rpm for 10 min to obtain the bacterial precipitate and the supernatant of the fermentation broth.

[0035] 3. Cell precipitation treatment: The cell precipitate and acidic alcohol solvent (chromatographic grade methanol, pH 3.5) were mixed at a mass ratio of 1:10, and subjected to ultrasonication (ultrasonic power of 300W, ultrasonic time of 10min), extraction (extraction at room temperature for 2h), and a second solid-liquid separation (centrifugation speed of 8000 rpm, centrifugation time of 10min) in sequence to obtain the cell precipitate and the supernatant containing styracil.

[0036] 4. Repeat the process: Repeat step 3 above until the bacteria turn purplish-gray. Combine the supernatants containing styraxin to obtain the first crude styraxin product.

[0037] 5. Concentration: The crude first lecithin was concentrated by rotary evaporation at a temperature of 40°C to obtain the crude second lecithin.

[0038] 6. Filtration: The crude second-grade erythromycin was filtered through a 0.45 μm filter membrane to obtain a sample ready for liquid preparation.

[0039] 7. Semi-preparative liquid chromatography separation: The filtered crude erythromycin was separated by semi-preparative liquid chromatography. The semi-preparative liquid chromatography included a manual injector, a UV detector, a C18 column, and a fraction collection zone. The detection wavelength was set to 535 nm, the flow rate was 4.72 mL / min, the sample loading volume was 5 mL, and isocratic elution mode was used. The elution process was isocratic elution, and the eluent was a mixture of an aqueous solution containing 0.1% (v / v) trifluoroacetic acid and chromatographic grade methanol at a v / v ratio of 35:65. The preparative liquid chromatography chromatogram is shown below. Figure 2 Real-time monitoring of the chromatographic peak; when the peak intensity reaches 200 mAU and the retention time is 35 min, the eluent begins to turn red. Collect the high-purity fraction of the target styraxin. Figure 3 .

[0040] 8. Rotary evaporation concentration: the prodigiosin fraction collection liquid is subjected to rotary evaporation concentration, the rotary evaporation concentration temperature is 30°C, and a prodigiosin flocculent state solution is obtained.

[0041] 9. Centrifugation: the prodigiosin flocculent state solution (see Figure 4 ) is subjected to centrifugation, the centrifugation speed is 8000 rpm, the centrifugation time is 10 min, the supernatant is discarded, and a prodigiosin precipitate is obtained.

[0042] 10. Drying: the prodigiosin precipitate is subjected to drying by a vacuum freeze dryer, and a powder prodigiosin ( Figure 1 ) is obtained.

[0043] After comparison with the standard curve of the PG standard (see Figure 5 , data in Table 1), the purity of the prodigiosin pure product separated and extracted from the fermentation broth by the method provided in this embodiment is > 99% (see Figure 6 , data in Tables 2 and 3), and the results of thin layer chromatography ( Figure 7 ) and LC-MS ( Figure 8 ) detection show that the purified product is PG.

[0044] Table 1 PG standard curve

[0045] Table 2 PG standard analysis liquid phase results

[0046] Table 3 PG self-extraction analysis liquid phase results

[0047] Experiment 1, condition optimization for detecting the purity of prodigiosin using an analytical liquid chromatograph The difference between this embodiment and the previous embodiment is that: The eluent is a mixed solution of 0.1% (by volume) trifluoroacetic acid aqueous solution and chromatographic grade methanol at a volume ratio of 30:70. The remaining steps are the same as in Example 1. The optimization of the elution conditions also reflects the stability and efficiency of the method: it is found that using a mixed solution of 0.1% (by volume) trifluoroacetic acid aqueous solution and chromatographic grade methanol at a volume ratio of 30:70 as the eluent can advance the peak time of prodigiosin, shorten the analysis time, and improve the overall efficiency. It is detected that the purity of the prodigiosin pure product separated and extracted from the fermentation broth by the method provided in this embodiment is > 99% (Table 4), and the peak time is advanced to about 12 min, the efficiency is improved, and see Figure 9 .

[0048] Table 4 Analysis liquid phase results after optimization of eluent conditions

[0049] Similarly, using a mixed solution of 0.1% (by volume) trifluoroacetic acid in water and chromatographically pure methanol at a volume ratio of 25:75 as the eluent, the peak time can be further advanced, and the peak time is advanced to about 6 min, and the efficiency is improved. These optimization options enable the method to maintain good stability and efficiency under different requirements. The sample analysis chart after purification, the overall optimized conditions, analysis time and retention time are as follows Figure 10 .

[0050] Experiment II, in vivo and in vitro biological activity experiment verification 1. Cell proliferation experiment 1.1 Experimental materials Self-extract PG: pyocyanin extracted by the present application.

[0051] Standard PG: commercially available high-purity pyocyanin (Aldrin, P274778).

[0052] Cell line: human nasopharyngeal carcinoma cell line (CNE2).

[0053] Culture medium: RPMI 1640 medium (serum-free medium: used for preparing drug solutions), 10% fetal bovine serum (FBS) complete medium, and 20% fetal bovine serum (FBS) complete medium.

[0054] CCK8 kit: for detecting cell viability.

[0055] 1.2 Experimental method 1.2.1 Cell culture and plating (1) Cell culture: CNE2 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and placed in a cell incubator at 37°C and 5% CO2 until the cells grew to the logarithmic phase.

[0056] (2) Cell counting and adjustment: count the cells under a microscope using a hemocytometer, and adjust the cell suspension concentration using complete medium containing 20% fetal bovine serum (FBS).

[0057] (3) Cell plating: add 50 μL of cell suspension to each well of a 96-well plate to make the cell density 5000 cells / well. Place the 96-well plate in a cell incubator at 37°C and 5% CO2 for 12 h to allow the cells to adhere.

[0058] 1.2.2 Drug treatment (1) Drug preparation: The self-extracted and dissolved 5 mM PG and 1 mg / mL PG standard were prepared into PG drug solutions with final concentrations of 50 nM, 100 nM, 200 nM, 400 nM, 800 nM and 1600 nM using serum-free culture medium.

[0059] (2) Drug treatment: 50 μL of PG drug solution with different concentrations was added to 96-well plates, with 3 replicates for each group. A blank control group (containing only serum-free culture medium) was also set up. The cells were cultured in a cell culture incubator at 37℃ and 5% CO2 for 48 h.

[0060] 1.2.3 CCK8 Detection (1) Reagent addition: Take out the 96-well plate after culturing and add 10 μL of CCK8 solution to each well.

[0061] (2) Incubation: Place the 96-well plate back into a cell culture incubator at 37°C and 5% CO2 for 1 hour.

[0062] (3) Absorbance measurement: The absorbance of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0063] 1.3. Experimental Results Absorbance values: The absorbance values ​​of CNE2 cells treated with different concentrations of self-extracted PG and PG standard at OD 450nm are shown below. Figure 11 That is, the inhibitory effects of the self-extracted PG and the PG standard on CNE2 cells are comparable, indicating that the strychnine extracted in this invention has cell proliferation inhibitory activity comparable to that of the standard.

[0064] 1.4 Conclusion The results of cell viability assay using the CCK8 assay showed that the squalene extracted in this invention (self-extracted PG) exhibited significant inhibitory activity against the human nasopharyngeal carcinoma cell line CNE2 in vitro, and its inhibitory effect was comparable to that of commercially available high-purity PG standards. This further demonstrates the high efficiency of the extraction method and the bioactivity of the extracted product, providing strong experimental evidence for further research and clinical application of squalene.

[0065] 2. Apoptosis experiment 2.1 Experimental Materials The PG material is the same as above.

[0066] Cell line: Human myeloma cell line (OCI-My5, suspension cells).

[0067] Annexin V-FITC / PI double staining kit.

[0068] 2.2 Experimental Methods (1) Cell culture: OCI-My5 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) in a cell incubator at 37°C, 5% CO2 until the cells grew to the logarithmic phase.

[0069] (2) Cell treatment: OCI-My5 cells: Logarithmic growth phase OCI-My5 cells were inoculated in a 12-well plate at a density of 1 x 10 5 cells per well, and serum-free medium was used for treatment.

[0070] (3) Drug treatment: Prepare PG standard and self-extracted PG solution at different concentrations (100 nM, 200 nM).

[0071] Add the above PG standard and self-extracted PG at different concentrations to OCI-My5 cells, with 3 replicates for each group. Continue to culture in a cell incubator at 37°C, 5% CO2 for 48h.

[0072] (4) Cell apoptosis detection - cell collection, resuspension and staining: OCI-My5 cells: Collect cells by centrifugation at 1000 rpm for 5 min.

[0073] Resuspend the cells with 100 μL of 1 x Binding Buffer, add 4 μL of Annexin V FITC, and incubate at room temperature for 15 min.

[0074] Add 2 μL of PI and incubate at room temperature for 3 min.

[0075] Add 400 μL of 1 x Binding Buffer to terminate the reaction.

[0076] (5) Flow cytometry detection: FITC Annexin V is excited by a 488 nm laser, and the fluorescence emission spectrum is detected at about 530 nm; the PI channel emits a spectrum at about 617 nm.

[0077] 2.3. Data analysis: FlowJo software was used to process flow cytometry data to analyze the apoptosis effect of PG standard and self-extracted PG on OCI-My5 cells.

[0078] 2.4 Experimental results The apoptosis rates of OCI-My5 cells treated with PG standard and self-extracted PG at different concentrations for 48h are as follows Figure 12The experimental results show that the total apoptosis rate of CNE2 cells induced by the self-extracted PG and the standard PG is not significantly different (P>0.05), which indicates that the extracted prodigiosin has the same cell apoptosis induction activity as the standard product.

[0079] 3. In vivo experiment 3.1 Experimental materials Self-extracted PG: prodigiosin extracted by the application.

[0080] Solvent: 2% DMSO + 8% methanol + 12% polyvinyl fluoride + 78% physiological saline solution.

[0081] Cell line: 5TGM1-LUC cells (used to establish a mouse multiple myeloma (MM) model).

[0082] Animal model: C57BL / KaLwRij mice (female, 16 weeks old, 20g~28g).

[0083] Experimental equipment: fluorescence imaging system (used to monitor tumor growth).

[0084] 3.2 Experimental method 3.2.1 Animal grouping and model establishment (1) Animal grouping: select C57BL / KaLwRij mice (female, 16 weeks old, 20g~28g), and randomly divide them into 2 groups, 4 in each group. The grouping is as follows: self-extracted PG treatment group, solvent treatment control group.

[0085] (2) Animal modeling: for the treatment group, 5TGM1-LUC cells are injected into the lateral tail vein of the mice at a inoculation amount of 1×10 6 cells / 200 μL per mouse, to establish a C57BL / KaLwRij mouse multiple myeloma (MM) cell allograft model. The control group of mice is also injected into the lateral tail vein, but the injection is of solvent without cells.

[0086] 3.2.2 Experimental scheme (1) Treatment cycle: start dosing one week after inoculation. The self-extracted PG treatment group of mice is injected intraperitoneally with a dose of 1.5 mg / kg of self-extracted PG (dissolved in 2% DMSO + 8% methanol + 12% polyvinyl fluoride + 78% physiological saline), once every two days. The control group of mice is injected intraperitoneally with the same volume of solvent as a control.

[0087] (2) Treatment duration: terminate the experiment after 5 weeks of medication.

[0088] 3.2.3 Observation of tumor-bearing conditions Fluorescence imaging: The growth of tumors in mice was monitored regularly using a fluorescence imaging system. Imaging was performed once a week, and changes in fluorescence intensity were recorded to assess tumor growth and metastasis.

[0089] 3.3 Experimental results 3.3.1 Tumor-bearing situation Fluorescence imaging results: The fluorescence intensity of mice in the self-extracted PG treatment group was significantly lower than that of the control group, indicating that self-extracted PG can effectively inhibit tumor growth and metastasis. The specific fluorescence intensity changes are shown in Figure 13

[0090] 3.4 Conclusion Through in vivo experiments, the extracted prodigiosin (self-extracted PG) of the present application showed significant anti-tumor activity in the C57BL / KaLwRij mouse multiple myeloma model, effectively inhibited tumor growth and metastasis, and had no significant adverse effects on the overall health of mice. This further demonstrates the high efficiency of the extraction method and the biological activity of the extracted product, providing strong experimental evidence for the clinical application of prodigiosin.

[0091] Comparative example: This comparative example provides a method for extracting prodigiosin from a fermentation broth containing prodigiosin. The difference from the example is that the semi-preparative liquid chromatography separation step is not performed, and the remaining steps are the same as the example.

[0092] After detection, the purity of the prodigiosin pure product separated and extracted from the fermentation broth using the method provided in the comparative example was 94.8%, as shown in Figure 14 (before purification), Table 5. The purity of the prodigiosin pure product separated and extracted in the example was 98.2%, as shown in Figure 14 (after purification), Table 6. It can be seen that the purification effect of the comparative example is much lower than that of the example.

[0093] Table 5 HPLC results of self-extracted PG before purification

[0094] Table 6 HPLC results of self-extracted PG after purification

[0095] ​Overall, the prior art mostly uses silica gel column chromatography combined with multi-step purification method, the steps are complex, the types and amount of organic solvents are large, and the obtained product has not been reported for in vivo and in vitro biological activity verification, and the practical application value needs to be clarified. The application provides a specific specification filter membrane filtration-semi-preparative HPLC-one-step refining method under the premise of maintaining direct sample feeding of the fermentation liquor, adopts a C18 chromatographic column, uses methanol-water (containing 0.1 % trifluoroacetic acid) as a single mobile phase, 4.72 mL / min, 535 nm online monitoring and automatic collection of the main peak, and after rotary evaporation, centrifugation and freeze-drying, prodigiosin is obtained, the single machine purification running time is less than or equal to 1.5 h, the purity is greater than or equal to 99 %, the total amount of organic solvents is only methanol and dilute TFA, the total amount is reduced by about 70 %, and no halogenated solvent is used, and the occupational exposure limit is relaxed. The application also carries out in vitro cytotoxicity test and in vivo tumor inhibition test on the high-purity product for the first time, and proves that the activity retention rate of prodigiosin extracted by the application is greater than or equal to 95 %. It can be seen that the method of the application provides a simple, stable and economical solution for the large-scale preparation of high-purity and high-activity prodigiosin.

Claims

1. A method for directly producing high-purity, high-activity prodigiosin from a fermentation broth, characterized by, The method comprises the following steps: (1) performing solid-liquid separation on a fermentation liquor containing prodigiosin to obtain a bacterial precipitate, mixing the bacterial precipitate with an acidic alcohol solvent, performing ultrasonic treatment, leaching, and solid-liquid separation to obtain a supernatant containing prodigiosin, and performing concentration to obtain a crude prodigiosin product; (2) filtering the crude prodigiosin product to obtain a sample solution, performing semi-preparative liquid chromatography separation after sample loading, performing elution using a mobile phase, monitoring chromatographic peaks in real time, and collecting a fraction of target prodigiosin according to signal intensity and / or color change of effluent; (3) performing concentration on the collected prodigiosin fraction, centrifuging to remove supernatant, obtaining a prodigiosin precipitate, and drying to obtain a powdered prodigiosin.

2. The method of claim 1, wherein, In step (1), the prodigiosin-containing fermentation broth is Serratia marcescens fermentation broth. Serratia marcescens ) fermentation broth.

3. The method of claim 2, wherein, The Serratia marcescens ( Serratia marcescens The preparation method of fermentation broth includes the following steps: inoculating plate-activated Serratia marcescens into seed culture medium, and waiting for the strain to grow to the logarithmic growth phase to obtain the inoculated strain; inoculating the inoculated strain into antibiotic-free LB liquid culture medium, and shaking culture in a shaker at a temperature of 27~31℃ and a speed of 150~170 rpm for 24~48h to obtain Serratia marcescens fermentation broth.

4. The method of claim 1, wherein, In step (1), the acidic alcohol solvent is chromatographic-grade methanol, the pH value is 3.0-4.0, and the mass ratio of the bacterial precipitate to the acidic alcohol solvent is 1:8-12.

5. The method of claim 1, wherein, In step (1), the ultrasonic treatment power is 250 W-350 W, the ultrasonic treatment time is 8 min-12 min; the leaching is performed at room temperature for 1.5 h-2.5 h; the centrifugal speed for solid-liquid separation is 7000 rpm-9000 rpm, and the centrifugal time is 8 min-12 min; the steps of ultrasonic treatment, leaching, and solid-liquid separation are repeated until the color of the bacterial precipitate becomes purple gray, and all supernatants are combined.

6. The method of claim 1, wherein, In step (1), the concentration is performed by rotary evaporation, and the rotary evaporation temperature is 40-45℃.

7. The method of claim 1, wherein, In step (2), the filter membrane used for filtration has a pore size of ≤0.45 μm.

8. The method of claim 1, wherein, In step (2), the semi-preparative liquid chromatography separation conditions include: using a C18 chromatographic column and an ultraviolet detector, setting the detection wavelength to 535 nm, setting the flow rate to 4.5-5.0 mL / min, setting the sample loading amount to 1-5 mL, using isocratic elution mode, and using a mixture of water containing 0.1% trifluoroacetic acid by volume and chromatographic-grade methanol at a volume ratio of 25-35:65-75 as the eluent.

9. The method of claim 1, wherein, In step (2), the collection of the fraction of target prodigiosin according to signal intensity and / or color change of effluent specifically includes: when the intensity of the chromatographic peak is 50-200 mAU, the retention time is 20-50 min, and / or when the color of the effluent starts to be red, the fraction of target prodigiosin is collected.

10. The method of claim 1, wherein, In step (2), the concentration is performed by rotary evaporation, and the rotary evaporation concentration temperature is 30-35℃.

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