Method for extracting stabilizer from blue-green algae

Abstract

The invention discloses a method for extracting a stable body from blue-green algae. The method comprises the following steps: S1, culturing the blue-green algae; s2, pretreating blue-green algae; s3, performing ultrasonic crushing; s4, performing differential centrifugation; s5, performing ultrafiltration; s6, carrying out ultracentrifugation; s7, density gradient centrifugation; and S8, removing peripheral proteins. The extraction method provided by the invention is high in extraction efficiency, stable and controllable in process, free of toxic and side products, green and environment-friendly, does not need high-temperature and high-pressure devices and large-scale instruments and equipment, expands the application scene, and reduces the application cost. The stable active particle biological material with high polyPn polymerization degree, high concentration and high purity can be obtained, the biological activity is high, the application requirements in the fields of food, daily chemicals and the like can be met, the strict requirements when the stable active particle biological material is applied to clinical medicine and scientific research are also met, and the stable active particle biological material has great practical application value and commercial prospects. The technical problem of preparing the stable active particle biological material in China is solved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for extracting a stable body from cyanobacteria. Background Art

[0002] Polyphosphates (polyP n ) is a long-chain polymer composed of 3 to 1000 phosphate (Pi) units connected by high-energy phosphate bonds. n PolyP is widely present in nature and has a high content. It is widely distributed in various biological cells in the form of particles, colloids or dissolved forms, including bacteria, fungi and higher mammals. It plays an important role in various organisms. n It is not only a storage reservoir of Pi and a source of energy for cell metabolism, but also has the function of chelating metal cations. n It plays a key role in biofilm formation, biological toxicity and stress response. In prokaryotes and some eukaryotic microorganisms, polyP n The synthesis of polyphosphate is catalyzed by polyphosphate kinases (PPKs), and most eukaryotic organisms synthesize polyP n The core mechanism of the transport of poly(P) molecules depends on the vacuolar transporter chaperone (VTC) complex, both of which use Pi at the end of ATP to synthesize poly(P n .polyP n The decomposition of polyphosphatase is catalyzed by enzymes such as exopolyphosphatase (PPX) and endopolyphosphatase (PPN).

[0003] The stabilosome is a subcellular structural unit that contains polyP n, phosphate Pi, metal cations, proteins, amino acids, lipids and water, etc., are widely present in most prokaryotes and some eukaryotic organisms. Under an optical microscope, they appear as dense black particles, so they are sometimes called electron-dense organelles, and various transmembrane proteins are distributed on the membranes of these particles. Different biologists have different perspectives and have different names for this subcellular structure containing granular polyphosphate. For example, this structure was originally separated from Trypanosoma and is called acidic calcium body (acidocalcisome). In algae, it is sometimes called polyphosphate granule (polyphosphate granule), and some scholars call it metachromatic granule, spirochete granule (volutin granule) and polyphosphate vacuole (polyphosphate vacuole), etc. According to its function in the organism, the present invention uniformly names it as stable body.

[0004] The stable body is not only a storage form of phosphorus, but also participates in a variety of biochemical reactions in cells, including DNA replication, repair and protein synthesis. The stable body is also related to cell signal transduction. It can regulate the concentration of calcium ions in cells and affect cell proliferation and differentiation. In some cases, the stable body may also participate in the stress response of cells, helping cells cope with changes in the external environment. In addition, researchers have found that the stable body can help treat diseases such as enteritis, which is of great significance for the further development of polyP n The ingredients of health products and medicines are of great significance.

[0005] In the current field of biomedical research, the in vivo separation and extraction technology of stable bodies has become a research hotspot. However, this technology faces a series of challenges, including cell disruption, release of stable bodies, separation and purification processes. These steps need to be carefully designed to avoid degradation or change of the stable body structure and reduce interference from other components in the organism. At present, the separation and extraction of stable bodies mainly rely on physical and chemical methods, including centrifugation, filtration and chromatography. Although these methods have certain limitations in extraction efficiency, purity and cost-effectiveness, they are still indispensable tools in current research. The gel chromatography method used by Guang et al. uses the difference in the size of stable body particles to achieve separation through a gel chromatography column. Although this method has a good separation effect, the required equipment is expensive, including high-performance liquid chromatography (HPLC) and precision detectors. The purchase cost of these equipment is high, which requires the laboratory to invest a lot of money. The gel chromatography column may fail due to the accumulation of impurities in the sample and needs to be replaced regularly to maintain the separation effect, further increasing the cost of using the chromatographic column. In addition, the particles and impurities in the sample can easily cause the chromatographic column to be blocked, affecting the separation efficiency and the reproducibility of the results. The chromatographic column needs to be maintained and cleaned regularly, which increases the complexity and cost of the operation. Gel chromatography has high requirements for laboratory environment, including temperature, humidity and dust-free environment. If the laboratory conditions are not up to standard, the separation effect and the service life of the equipment may be affected. Therefore, the above problems of gel chromatography limit its feasibility in large-scale applications. The density gradient centrifugation method proposed by Huang et al. uses the gradient formed by different density media (such as iodixanol and sucrose, etc.) to separate stable body particles through centrifugal force. Although this method can effectively separate particles of different densities, the purity of the separated stable body is relatively low, and it is easy to mix with other cell fragments and impurities. In addition, during the density gradient centrifugation process, cells are easy to rupture, resulting in cytoplasmic contamination of the separated stable body particles, which reduces the purity and activity of the stable body. In addition, the ultrafiltration method proposed in patent CN115678832 A realizes the separation and purification of exosome particles through ultrafiltration membranes of different pore sizes. It has the advantages of simple operation and suitability for large-scale separation, but it is difficult to separate the internal biological particles in the broken algae liquid. Patent CN118028215 A proposes a method for extracting exosomes by ultracentrifugation. Although this method can quickly separate particles, the purity of the separated exosomes is not high, and it is usually necessary to combine it with other separation methods to improve the purity. Although the existing separation and extraction technologies have their own advantages, in order to obtain high-purity stable bodies, it is still necessary to further explore and optimize these methods, or develop new technical strategies to overcome the limitations of existing technologies.

[0006] Natural stabilizers can be used in the fields of medicine, food, daily chemicals and agriculture. Applications in the medical field include: as a component of the drug delivery system, the nanometer size and high permeability of the stabilizer are used to improve the distribution and absorption of drugs in the body; as a vaccine adjuvant, it can enhance the immune response and improve the efficacy of the vaccine; in regenerative medicine, it is used as a bioactive material to promote cell proliferation and tissue repair. In the food field, stabilizers can be used to develop functional foods with specific health benefits; they can be used as food additives to enhance the nutritional value of food or improve the preservation properties of food. In the field of daily chemicals, stabilizers can be used as active ingredients to improve skin health and appearance due to their bioactivity and nanometer size; in oral care products, they can be used for antibacterial and anti-inflammatory effects to promote oral tissue health. In agricultural applications, they can be used as biostimulants to promote crop growth and improve crop disease resistance and stress resistance; as a component of pesticides, their biological activity is used to control pests and pathogens. Therefore, stabilizers are in high demand in the market, but a method for extracting high-purity, pollution-free biosource stabilizers has not yet been established. Summary of the invention

[0007] The present invention aims to solve the problem of low purity of stabilizers in existing stabilizer extraction methods. The stabilizers extracted by existing technologies contain more impurities and by-products, which not only affects its safety and effectiveness in clinical treatment, but also limits its application potential in the field of scientific research. The purpose of the present invention is to provide a stabilizer extraction method that can extract high-purity stabilizers, has low health and environmental risks, and has wide adaptability. The extracted stabilizer particles have high purity, good uniformity, and strong activity, which can not only meet the application requirements in the fields of food, daily chemicals, etc., but also meet the strict requirements when applied to clinical medicine and scientific research.

[0008] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0009] A method for extracting a stable body from cyanobacteria comprises the following steps:

[0010] S1. Cultivation of cyanobacteria: Cyanobacteria were inoculated into a blue-green algae culture medium (BG11 culture medium) at a ratio of 10% v / v, solid-liquid separation was performed, and cyanobacteria were collected;

[0011] S2. Cyanobacteria pretreatment: washing the cyanobacteria obtained in step S1, separating the solid from the liquid, and collecting the cyanobacteria;

[0012] S3. Ultrasonic disruption: resuspend the cyanobacteria obtained in step S2 in a saline solution isotonic with the cyanobacteria, and ultrasonically disrupt the cyanobacteria to obtain a cyanobacterial homogenate;

[0013] S4. Differential centrifugation: The cyanobacteria homogenate obtained in step S3 is subjected to differential centrifugation to obtain a preliminary separation solution; large particles of impurities, including dead cells, EPS and other impurities, are removed from the cyanobacteria homogenate obtained in step S3 by differential centrifugation in step S4;

[0014] S5. Ultrafiltration: The preliminary separation liquid obtained in step S4 is filtered and ultrafiltered, and the concentrated liquid is collected to obtain a crude extract of the stable body; impurities larger than 0.22 μm in the preliminary separation liquid obtained in step S4 are removed by filtering in step S5, and impurities with a molecular weight less than 100 kDa are removed by ultrafiltration, leaving the stable body particles and some other particulate impurities, to obtain a crude extract of the stable body, which contains more impurities (cell membranes, pigment particles, etc.).

[0015] A method for extracting a stable body from cyanobacteria, further comprising the following steps:

[0016] S6. Ultracentrifugation: The concentrate obtained in step S5 was resuspended in a NaCl aqueous solution or a buffer solution in a volume ratio of 1:5 to 10, and ultracentrifuged. The precipitate was resuspended in an aqueous solution of iodixanol to obtain a resuspended solution; the nano-sized particles (including the stable body particles) in the crude extract of the stable body were further enriched by ultracentrifugation in step S6;

[0017] S7. Density gradient centrifugation: adding iodixanol aqueous solutions with concentrations of 50% w / v, 45% w / v, 40% w / v, 35% w / v, 30% w / v, 25% w / v, 20% w / v, 15% w / v, 10% w / v, 8% w / v, 5% w / v and 2% w / v to an ultracentrifuge tube in descending order of concentration, adding the resuspension obtained in step S6 to the ultracentrifuge tube, performing ultracentrifugation, and collecting an iodixanol suspension with a concentration of 10% w / v; further removing residual macromolecular non-stable particles and residual small-size cell membranes in the crude stable body extract by density gradient centrifugation in step S7;

[0018] S8. Removal of peripheral proteins: The 10% w / v iodixanol suspension obtained in step S7 is mixed with Tween20, shaken at room temperature, and ultrafiltered. The obtained ultrafiltration concentrate is resuspended with NaCl aqueous solution or buffer, ultrafiltered, and the concentrate is collected to obtain a high-purity stable body extract; step S8 eliminates the residual cytoplasmic proteins in the stable body crude extract by Tween20 to further improve the protein purity of the stable body extract.

[0019] In step S1, the culture conditions are 50-60°C, a light-dark ratio of 12h:12h, a light intensity of 2000-4000lx, and a culture period of 14-28 days; the solid-liquid separation is centrifugation; the centrifugation conditions are 3000-8000g for 10-20min, preferably 6000g for 10min.

[0020] In step S2, the washing is performed using a NaCl aqueous solution or a PBS buffer solution, preferably, the washing is performed using a 0.15 mM NaCl aqueous solution or a PBS buffer solution with a concentration of 5 to 10 mM and a pH of 7.2 to 7.4; the solid-liquid separation is performed by centrifugation; the centrifugation is performed under conditions of 3000 to 8000 g for 10 to 20 min, preferably 6000 g for 10 min; the washing and the solid-liquid separation operations are repeated 1 to 10 times, preferably 3 to 5 times; when the solid-liquid separation is performed by centrifugation, the washing and the centrifugation are performed simultaneously in a centrifuge tube.

[0021] In step S3, the salt solution isotonic with the cyanobacteria is a NaCl aqueous solution, preferably a NaCl aqueous solution with a concentration of 0.15 mM; the ultrasound has a frequency of 20 to 25 kHz and an ultrasound time of 5 to 10 min, preferably a frequency of 25 kHz and a time of 10 min.

[0022] In step S4, the differential centrifugation is performed under the following conditions: the cyanobacteria homogenate obtained in step S3 is centrifuged at 300-600 g for 10-20 min, the supernatant is collected, the cyanobacteria homogenate is centrifuged at 1000-3000 g for 10-20 min, the supernatant is collected, and the supernatant is collected by centrifugation at 6000-12000 g for 20-40 min. Preferably, the conditions are as follows: the cyanobacteria homogenate obtained in step S3 is centrifuged at 300 g for 10 min, the supernatant is collected, the supernatant is collected by centrifugation at 2000 g for 10 min, the supernatant is collected, and the supernatant is collected by centrifugation at 10000 g for 30 min.

[0023] In step S5, the filtration is water filter membrane filtration, preferably, filtration through 0.45 μm and 0.22 μm water filter membranes; the ultrafiltration has an ultrafiltration membrane with a molecular weight cutoff of 100 kDa.

[0024] In step S6, the NaCl aqueous solution is a 0.15mM NaCl aqueous solution, and the buffer is a PBS buffer with a concentration of 5-10mM and a pH of 7.2-7.4; the ultracentrifugation is performed at a temperature of 4-10°C, a centrifugal force of 120,000-150,000g, and a centrifugation time of 1-2h, preferably at a temperature of 4°C, a centrifugal force of 150,000g, and a centrifugation time of 1.5h; the concentration of the iodixanol aqueous solution is 45-55% w / v, preferably 45% w / v.

[0025] In step S7, the ultracentrifugation is performed at a temperature of 4 to 10° C., a centrifugal force of 120,000 to 150,000 g, and a centrifugal time of 1 to 2 h. Preferably, the temperature is 4° C., the centrifugal force is 150,000 g, and the centrifugal time is 2 h. In the ultracentrifuge tube, the added amounts of the iodixanol aqueous solutions of different concentrations are the same. The added amount of the resuspension obtained in step S6 is the same as the added amount of the iodixanol aqueous solutions of each concentration.

[0026] In step S8, the 10% w / v iodixanol suspension obtained in step S7 is mixed with 0.3% v / v Tween20 in a volume ratio of 1:1-2; the room temperature oscillation is performed for 10-20 min, preferably 20 min; the ultrafiltration is performed with an ultrafiltration membrane having a molecular weight cutoff of 100 kDa; the NaCl aqueous solution is a 100 mM NaCl aqueous solution, and the buffer is a PBS buffer with a concentration of 5-10 mM and a pH of 7.2-7.4; and the operations of resuspending the ultrafiltration concentrate with the NaCl aqueous solution or the buffer, ultrafiltration, and collecting the concentrate are repeated 1-10 times, preferably 2-4 times.

[0027] In the present invention, the w / v is a mass-to-volume ratio, i.e. g / mL. For example, a 2% w / v iodixanol aqueous solution is an iodixanol aqueous solution with a concentration of 2 g / 100 mL.

[0028] Beneficial effects:

[0029] 1. The extraction method provided by the present invention has high extraction efficiency, and the obtained stable extract has high purity. The process of the method is stable and controllable, no toxic byproducts are produced, it is green and environmentally friendly, and no high temperature and high pressure device and large-scale instruments and equipment are required, which expands the application scenarios and reduces the application cost. It will solve the technical difficulties in preparing stable active granular biomaterials in China.

[0030] 2. The present invention can obtain high polyP n Stable active granular biomaterials with high polymerization degree, high concentration and high purity, polyP n The degree of polymerization can reach 150 or above. The particle concentration of the stable extract obtained by the present invention is calculated by nanoparticle tracking analysis (NTA) data to be 8.35×10 12 particles / L, and the granule protein concentration was 7.872×10 5 μg / L, the particle-protein ratio is 1.061×10 7 particles / μg, with very high purity and concentration and high biological activity. It can not only meet the application requirements in the fields of food and daily chemicals, but also meet the strict requirements for application in clinical medicine and scientific research. It has great practical application value and commercial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0032] Figure 1 This is the toluidine blue staining picture of the stable body in hot spring cyanobacteria;

[0033] Figure 2 PolyP in hot spring cyanobacteria n Electropherogram of chain length;

[0034] Figure 3 This is a sample image obtained after density gradient centrifugation in Example 1;

[0035] Figure 4 This is a toluidine blue staining image of the stable body in the sample obtained after density gradient centrifugation in Example 1. DETAILED DESCRIPTION

[0036] The present invention is further described below according to the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention. The specific techniques or conditions not specified in the examples are carried out according to the techniques or conditions described in the literature in this field, or according to the product instructions. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0037] In the following examples, the BG11 culture medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., and its formula was 1.50 g / L sodium nitrate, 0.04 g / L potassium hydrogen phosphate trihydrate, 0.074 g / L magnesium sulfate heptahydrate, 0.036 g / L calcium chloride dihydrate, 0.006 g / L citric acid, 0.006 g / L ammonium ferric citrate, 0.001 g / L EDTA, 0.02 g / L sodium carbonate, 0.00286 g / L boric acid, 0.00181 g / L manganese chloride monohydrate, 0.000222 g / L zinc sulfate heptahydrate, 0.000079 g / L copper sulfate pentahydrate, 0.00039 g / L sodium molybdate dihydrate, and 0.000049 g / L cobalt nitrate hexahydrate.

[0038] In the following examples, the iodixanol aqueous solutions with mass concentrations of 50% w / v, 45% w / v, 40% w / v, 35% w / v, 30% w / v, 25% w / v, 20% w / v, 15% w / v, 10% w / v, 8% w / v, 5% w / v and 2% w / v were prepared by diluting a 60% w / v iodixanol aqueous solution (OptiDensity purchased from Nanjing Jiyi Biotechnology Co., Ltd.) with pure water. TM Iodixanol solution, 60% w / v).

[0039] In the following embodiments, the particle concentration refers to the number of stable body particles in 1L of stable body extract, and the unit is "particles / L". The higher the particle concentration, the higher the concentration of stable body in the extract; the particle protein concentration refers to the content of total protein in 1L of stable body extract, and the unit is "μg / L"; the calculation formula of the particle protein ratio is: particle protein ratio = particle concentration / particle protein concentration, and the unit is "particles / μg". The larger the particle protein ratio, the higher the purity of the stable extract in the extract.

[0040] The biological source of the stable body in the following examples and comparative examples is hot spring cyanobacteria, which is obtained by separation and purification from algae samples collected in the wild. The specific method is as follows: 1. Collection of algae samples: Collect algae samples from wild water bodies and store them in sample bottles. 2. Separation of single algae cells: Take a small amount of algae sample and use a steel needle or a rubber-tipped dropper to break up the algae colony and separate single algae cells. 3. Centrifugation and dispersion: Transfer the separated single algae cells to a centrifuge tube and centrifuge to remove excess solution and impurities; after centrifugation, resuspend the algae cells to ensure that they are evenly dispersed in the solution. 4. Plate streaking and initial coating: Evenly spread the BG11 culture medium containing agar on the culture plate, use an inoculation loop to take out a small amount of the dispersed algae cell solution, use the streaking method to perform the initial coating on the culture medium, separate the colonies, and form circular colonies after cultivation. 5. Transfer and re-streaking of colonies: Use an inoculation loop to pick out a single cyanobacterial colony from the circular colonies produced by the initial coating, transfer it to a new BG11 medium containing agar, repeat the coating and streaking steps, and repeat this step at least three times to ensure that pure single-cell colonies are obtained. 6. Multi-well plate culture: Select the purified single-cell colonies and transfer them to a multi-well plate for culture. Each well in the multi-well plate contains an appropriate amount of BG11 medium to further isolate and culture single algae cells. 7. Amplification culture: Transfer the successfully isolated single-cell colonies to BG11 medium for large-scale amplification culture until enough cyanobacterial cells are obtained for subsequent extraction.

[0041] The optimum survival temperature of hot spring cyanobacteria is 45-50℃. Figure 1 This is a toluidine blue staining image of stable bodies in hot spring cyanobacteria. The black particles pointed by the red arrows in the image are stable bodies. It can be seen from the image that hot spring cyanobacteria are rich in stable body particles. Figure 2 PolyP in hot spring cyanobacteria n The electrophoresis diagram of the chain length shows that the long-chain polyP n With a higher degree of polymerization, polyP n The higher the degree of polymerization, the less likely the stable body is to decompose and the more stable it is.

[0042] Example 1

[0043] A method for extracting a high-purity stable body from hot spring cyanobacteria, the extraction method comprising the following steps:

[0044] S1. Cultivation of hot spring cyanobacteria: Inoculate the hot spring cyanobacteria into BG11 medium at a ratio of 10% v / v, and culture for 14 days at a constant temperature of 50°C, a light-dark ratio of 12h:12h, and a light intensity of 3000lx. After the cultivation, collect the algae solution (300mL) and put it into 6 50mL centrifuge tubes, centrifuge at 6000g for 10min to enrich the cyanobacteria, and collect the cyanobacterial precipitate.

[0045] S2. Cyanobacteria pretreatment: The cyanobacteria obtained in step S1 were resuspended in 0.15 mM NaCl aqueous solution in 6 50 mL centrifuge tubes. Centrifuged at 6000 g for 10 minutes using a high-speed centrifuge, and the supernatant was removed. The washing was repeated four times, and the cyanobacteria precipitate was finally collected.

[0046] S3. Ultrasonic disruption: The cyanobacteria precipitate obtained in step S2 was resuspended in 0.15 mM NaCl aqueous solution in 6 50 mL centrifuge tubes, and ultrasonically disrupted at 25 kHz for 10 min to obtain 6 tubes of cyanobacteria homogenate;

[0047] S4. Differential centrifugation: The cyanobacteria homogenate obtained in step 3 was separated using a high-speed centrifuge. First, centrifuge at 300g for 10 minutes, take the supernatant, then centrifuge at 2000g for 10 minutes, take the supernatant again, and finally centrifuge at 10000g for 30 minutes, take the supernatant, and finally obtain 6 tubes of preliminary separation liquid.

[0048] S5. Ultrafiltration: The 6 tubes of preliminary separation liquid obtained in step S4 were first filtered through a 0.45 μm water system membrane, then filtered through a 0.22 μm water system membrane, and finally ultrafiltered using a 100 kDa ultrafiltration tube to collect the concentrated liquid, obtaining 200 μL of concentrated liquid in each tube, and finally the concentrated liquids were enriched together to a total of 1.2 mL.

[0049] S6. Ultracentrifugation: The 1.2 mL concentrate obtained in step S5 was resuspended with 10 mL of 0.15 mM NaCl aqueous solution, and ultracentrifuged at 150,000 g for 1.5 h at 4 ° C using an ultracentrifuge. The precipitate was then resuspended in 2 mL of 45% w / v iodixanol aqueous solution to obtain a resuspension.

[0050] S7. Density gradient centrifugation: 2 mL of 50% w / v, 45% w / v, 40% w / v, 35% w / v, 30% w / v, 25% w / v, 20% w / v, 15% w / v, 10% w / v, 8% w / v, 5% w / v and 2% w / v iodixanol aqueous solutions were added to an ultracentrifuge tube in descending order of concentration, and then 2 mL of the 45% w / v iodixanol resuspension obtained in step 6 was added to an ultracentrifuge tube, and ultracentrifuged at 150,000 g for 2 h at 4°C, and then 2 mL of the 10% w / v iodixanol suspension was collected. Figure 3 This is a sample obtained after density gradient centrifugation in this embodiment. It can be seen from the figure that density gradient centrifugation can well concentrate and separate the stable body from other cyanobacteria components; Figure 4 This is a toluidine blue staining image of the stable body in the sample obtained after density gradient centrifugation in this example, and the red arrow in the figure points to the stable body particles.

[0051] S8. Removal of peripheral proteins: Add 3 mL of 0.3% (v / v) Tween 20 to 2 mL of 10% w / v iodixanol aqueous solution obtained in step S7, shake at room temperature for 20 minutes, and then ultrafilter through a 100 kDa ultrafiltration tube to remove Tween 20. The resulting concentrate is resuspended in 1 mL of 100 mM NaCl aqueous solution, ultrafiltered through a 100 kDa ultrafiltration tube, and the concentrate is collected. The resulting concentrate is resuspended again in 1 mL of 100 mM NaCl solution, and finally ultrafiltered through a 100 kDa ultrafiltration tube to collect the concentrate, thereby obtaining a high-concentration, high-purity stable active particle extract.

[0052] Through the above steps, a high-concentration, high-purity stable extract can be obtained. n The degree of polymerization can reach 150 and above. The particle concentration in the stable body extract calculated by NTA data is 8.35×10 12 particles / L, and the granule protein concentration was 7.872×10 5 μg / L, the particle-protein ratio is 1.061×10 7 Particles / μg, the purity and concentration of the stable extract are very high and can be used in scientific research and medical fields.

[0053] Example 2

[0054] A method for extracting a stable body from hot spring cyanobacteria, the extraction method comprising the following steps:

[0055] S1. Cultivation of hot spring cyanobacteria: Inoculate the hot spring cyanobacteria into BG11 medium at a ratio of 10% v / v, and culture for 14 days at a constant temperature of 50°C, a light-dark ratio of 12h:12h, and a light intensity of 3000lx. After the cultivation, collect the algae solution (300mL) and put it into 6 50mL centrifuge tubes, centrifuge at 6000g for 10min to enrich the cyanobacteria, and collect the cyanobacterial precipitate.

[0056] S2. Cyanobacteria pretreatment: The cyanobacteria obtained in step S1 were resuspended in 0.15 mM NaCl aqueous solution in 6 50 mL centrifuge tubes. Centrifuged at 6000 g for 10 minutes using a high-speed centrifuge, and the supernatant was removed. The washing was repeated four times, and the cyanobacteria precipitate was finally collected.

[0057] S3. Ultrasonic disruption: The cyanobacteria precipitate obtained in step S2 was resuspended in 0.15 mM NaCl aqueous solution in 6 50 mL centrifuge tubes, and ultrasonically disrupted at 25 kHz for 10 min to obtain 6 tubes of cyanobacteria homogenate;

[0058] S4. Differential centrifugation: The cyanobacteria homogenate obtained in step 3 was separated using a high-speed centrifuge. First, centrifuge at 300g for 10 minutes, take the supernatant, then centrifuge at 2000g for 10 minutes, take the supernatant again, and finally centrifuge at 10000g for 30 minutes, take the supernatant, and finally obtain 6 tubes of preliminary separation liquid.

[0059] S5. Ultrafiltration: The 6 tubes of preliminary separation liquid obtained in step S4 were first filtered through a 0.45 μm water system membrane, then filtered through a 0.22 μm water system membrane, and finally ultrafiltered using a 100 kDa ultrafiltration tube to collect the concentrated liquid, obtaining 200 μL of concentrated liquid in each tube, and finally the concentrated liquids were enriched together to a total of 1.2 mL.

[0060] The particle concentration of the stable extract obtained by the above steps is 9.35×10 11 particles / L, and the particle protein concentration was 1.856×10 7 μg / L, the particle-protein ratio is 5.038×10 4 The concentration and purity of the stable body extract are lower than those of the stable body extract obtained in Example 1, and the stable body extract can be applied to food, daily chemicals, agriculture and other industries that do not require high purity of the stable body extract.

[0061] Comparative Example 3

[0062] A method for extracting a stable body from hot spring cyanobacteria, the extraction method comprising the following steps:

[0063] S1. Cultivation of hot spring cyanobacteria: Inoculate the hot spring cyanobacteria into BG11 medium at a ratio of 10% v / v, and culture for 14 days at a constant temperature of 50°C, a light-dark ratio of 12h:12h, and a light intensity of 3000lx. After the cultivation, collect the algae solution (300mL) and put it into 6 50mL centrifuge tubes, centrifuge at 6000g for 10min to enrich the cyanobacteria, and collect the cyanobacterial precipitate.

[0064] S2. Cyanobacteria pretreatment: The cyanobacteria obtained in step S1 were resuspended in 0.15 mM NaCl aqueous solution in 6 50 mL centrifuge tubes. Centrifuged at 6000 g for 10 minutes using a high-speed centrifuge, and the supernatant was removed. The washing was repeated four times, and the cyanobacteria precipitate was finally collected.

[0065] S3. Ultrasonic disruption: The cyanobacteria precipitate obtained in step S2 was resuspended in 0.15 mM NaCl aqueous solution in 6 50 mL centrifuge tubes, and ultrasonically disrupted at 25 kHz for 10 min to obtain 6 tubes of cyanobacteria homogenate;

[0066] S4. Differential centrifugation: The cyanobacteria homogenate obtained in step 3 was separated using a high-speed centrifuge. First, centrifuge at 300g for 10 minutes, take the supernatant, then centrifuge at 2000g for 10 minutes, take the supernatant again, and finally centrifuge at 10000g for 30 minutes, take the supernatant, and finally obtain 6 tubes of preliminary separation liquid.

[0067] S5. Ultrafiltration: The 6 tubes of preliminary separation liquid obtained in step S4 were first filtered through a 0.45 μm water system membrane, then filtered through a 0.22 μm water system membrane, and finally ultrafiltered using a 100 kDa ultrafiltration tube to collect the concentrated liquid, obtaining 200 μL of concentrated liquid in each tube, and finally the concentrated liquids were enriched together to a total of 1.2 mL.

[0068] S6. Ultracentrifugation: The 1.2 mL concentrate obtained in step S5 was resuspended with 10 mL of 0.15 mM NaCl aqueous solution, and ultracentrifuged at 150,000 g for 1.5 h at 4 ° C using an ultracentrifuge. The precipitate was then resuspended in 2 mL of 45% w / v iodixanol aqueous solution to obtain a resuspension.

[0069] S7. Density gradient centrifugation: 2 mL of iodixanol aqueous solutions with concentrations of 50% w / v, 45% w / v, 40% w / v, 35% w / v, 30% w / v, 25% w / v, 20% w / v, 15% w / v, 10% w / v, 8% w / v, 5% w / v and 2% w / v were added to an ultracentrifuge tube in descending order of concentration, and then 2 mL of the 45% w / v iodixanol resuspension obtained in step 6 was added to an ultracentrifuge tube, and ultracentrifuged at 150,000 g for 2 h at 4°C, and then 2 mL of the 10% w / v iodixanol suspension containing stable body particles was collected.

[0070] The granule protein ratio of the stable granule extract obtained by the above steps was 1.534×10 6 Compared with Example 3, Example 1 did not use TWeen20 to remove the cytoplasmic protein in the stable extract, and the stable granule protein ratio obtained in Example 3 was 1.534×10 6 In Example 1, the cytoplasmic protein in the stable extract was removed by TWeen20, and the particle-protein ratio was 1.061×10 7 The present invention uses 0.3% (v / v) TWeen20 to remove peripheral proteins in the stable body extract to obtain a higher purity stable body extract.

[0071] The present invention breaks through the limitations of existing methods in terms of the purity of the stable body extraction through innovative extraction technology, and provides a stable body extraction scheme that can obtain high-purity stable bodies, is safe, economical and environmentally friendly, to meet the urgent needs of clinical and scientific research for high-standard stable bodies. This technology can not only effectively remove impurities and by-products, enhance the biological activity of the stable body, and ensure the strict standards of clinical and scientific research applications, but also has a wide range of adaptability and is suitable for different PolyP n The polymerization degree and the stable body of biological origin have promoted innovative research in the fields of biomedicine, materials science and environmental science. In addition, the invention also supports the development of clinical treatment plans, improves the quality of life of patients, and promotes the development of related industries, including biomedicine, health products and environmental governance, opening up new opportunities and breakthroughs for clinical treatment and scientific research.

[0072] The present invention provides a method and idea for extracting a stable body from cyanobacteria. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for extracting a stable substance from cyanobacteria, characterized in that: The steps include: S1. Cultivation of cyanobacteria: inoculating cyanobacteria into a blue-green algae culture medium, culturing, solid-liquid separation, and collecting cyanobacteria; S2. Cyanobacteria pretreatment: washing the cyanobacteria obtained in step S1, separating the solid from the liquid, and collecting the cyanobacteria; S3. Ultrasonic disruption: resuspend the cyanobacteria obtained in step S2 in a saline solution isotonic with the cyanobacteria, and ultrasonically disrupt the cyanobacteria to obtain a cyanobacterial homogenate; S4. Differential centrifugation: The cyanobacteria homogenate obtained in step S3 was subjected to differential centrifugation to obtain a preliminary separation solution; S5. Ultrafiltration: The preliminary separation liquid obtained in step S4 is filtered and ultrafiltered, and the concentrated liquid is collected to obtain a stable crude extract.

2. The method according to claim 1, characterized in that The following steps are also included: S6 ultracentrifugation: The concentrate obtained in step S5 was resuspended in an aqueous NaCl solution or buffer, ultracentrifuged, and the precipitate was resuspended in an aqueous iodixanol solution to obtain a resuspended solution; S7. Density gradient centrifugation: Aqueous solutions of iodixanol having concentrations of 50% w / v, 45% w / v, 40% w / v, 35% w / v, 30% w / v, 25% w / v, 20% w / v, 15% w / v, 10% w / v, 8% w / v, 5% w / v and 2% w / v were added to an ultracentrifuge tube in descending order of concentration, and the resuspension obtained in step S6 was added to the ultracentrifuge tube, and ultracentrifuged to collect a 10% w / v iodixanol suspension; S8. Removal of peripheral proteins: The 10% w / v iodixanol suspension obtained in step S7 is mixed with Tween 20, shaken at room temperature, and ultrafiltered. The obtained ultrafiltration concentrate is resuspended with a NaCl aqueous solution or a buffer solution, ultrafiltered, and the concentrate is collected to obtain a high-purity stable body extract.

3. The method according to claim 1 or 2, characterized in that: In step S1, the culture conditions are 50-60°C, a light-dark ratio of 12h:12h, a light intensity of 2000-4000lx, and a culture period of 14-28 days; the solid-liquid separation is centrifugation; and the centrifugation conditions are 3000-8000g for 10-30min.

4. The method according to claim 1 or 2, characterized in that: In step S2, the washing is performed using a NaCl aqueous solution or a PBS buffer solution, preferably, the washing is performed using a 0.15 mM NaCl aqueous solution or a PBS buffer solution with a concentration of 5 to 10 mM and a pH of 7.2 to 7.4; the solid-liquid separation is performed by centrifugation; the centrifugation is performed under the conditions of 3000 to 8000 g for 10 to 20 min; the washing and the solid-liquid separation operations are repeated 1 to 10 times; when the solid-liquid separation is performed by centrifugation, the washing and the centrifugation are performed simultaneously in a centrifuge tube.

5. The method according to claim 1 or 2, characterized in that: In step S3, the salt solution isotonic with the cyanobacteria is a NaCl aqueous solution, preferably a NaCl aqueous solution with a concentration of 0.15 mM; the ultrasound has a frequency of 20 to 25 kHz and an ultrasound time of 5 to 10 min.

6. The method according to claim 1 or 2, characterized in that: In step S4, the differential centrifugation is performed under the following conditions: the cyanobacteria homogenate obtained in step S3 is centrifuged at 300-600 g for 10-20 min, the supernatant is collected, the cyanobacteria homogenate is centrifuged at 1000-3000 g for 10-20 min, the supernatant is collected, and the supernatant is collected after centrifugation at 6000-12000 g for 20-40 min.

7. The method according to claim 1 or 2, characterized in that: In step S5, the filtration is water filter membrane filtration, preferably, filtration through 0.45 μm and 0.22 μm water filter membranes; the ultrafiltration has an ultrafiltration membrane with a molecular weight cutoff of 100 kDa.

8. The method according to claim 2, characterized in that: In step S6, the NaCl aqueous solution is a 0.15mM NaCl aqueous solution; the buffer solution is a PBS buffer solution with a concentration of 5-10mM and a pH of 7.2-7.4; the ultracentrifugation temperature is 4-10°C, the centrifugal force is 120000-150000g, and the centrifugation time is 1-2h; the concentration of the iodixanol aqueous solution is 45-55% w / v.

9. The method according to claim 2, characterized in that: In step S7, the ultracentrifugation temperature is 4-10° C., the centrifugal force is 120,000-150,000 g, and the centrifugation time is 1-2 h; in the ultracentrifuge tube, the added amounts of the iodixanol aqueous solutions of different concentrations are the same; and the added amount of the resuspension obtained in step S6 is the same as the added amount of the iodixanol aqueous solutions of each concentration.

10. The method according to claim 2, characterized in that In step S8, the 10% w / v iodixanol suspension obtained in step S7 is mixed with 0.3% v / v Tween20 in a volume ratio of 1:1-2; the room temperature oscillation is performed for 10-20 minutes; the ultrafiltration is performed with an ultrafiltration membrane having a molecular weight cutoff of 100 kDa; the NaCl aqueous solution is a 100 mM NaCl aqueous solution; the buffer is a PBS buffer with a concentration of 5-10 mM and a pH of 7.2-7.4; and the operations of resuspending the ultrafiltration concentrate with the NaCl aqueous solution or buffer, ultrafiltration, and collecting the concentrate are repeated 1-10 times.

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