A method for extracting stable bodies from cyanobacteria

Abstract

The present invention discloses a method for extracting a stable body from cyanobacteria, comprising the following steps: S1. Cultivating cyanobacteria; S2. Pretreating cyanobacteria; S3. Ultrasonic disruption; S4. Differential centrifugation; S5. Ultrafiltration; S6. Ultracentrifugation; S7. Density gradient centrifugation; S8. Removing peripheral proteins. The extraction method provided by the present invention has high extraction efficiency, stable and controllable process, no toxic by-products, is green and environmentally friendly, and does not require high temperature and high pressure equipment and large instruments and equipment, thereby expanding application scenarios and reducing application costs. The present invention can obtain high polyP n Stable, active granular biomaterials with high polymerization degree, concentration, and purity, as well as high bioactivity, can meet the stringent requirements of applications in the food and daily chemical sectors, as well as clinical medicine and scientific research. They possess significant practical application value and commercial prospects, and will address the technical challenges of preparing stable, active granular biomaterials in China.

Description

Technical Field

[0001] The present 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 dissolution, 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 kinase (PPKs), and most eukaryotic organisms synthesize polyP n The core mechanism of the transport of poly(P) is dependent 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 phosphatase is catalyzed by enzymes such as exopolyphosphatase (PPX) and endopolyphosphatase (PPN).

[0003] Stable bodies are subcellular structural units containing polyP n, phosphate Pi, metal cations, as well as proteins, amino acids, lipids and water, are widely present in most prokaryotes and some eukaryotic organisms. Under an optical microscope, they appear as dense black particles, and are therefore sometimes referred to as electron-dense organelles. Various transmembrane proteins are distributed on the membranes of these particles. Different biologists have different perspectives and have named this subcellular structure containing granular polyphosphate differently. For example, this structure was originally isolated from Trypanosoma and is called an acidic calcium body. In algae, it is sometimes called a polyphosphate granule. Some scholars also call it metachromatic granules, volutin granules and polyphosphate vacuoles. According to its function in the organism, the present invention uniformly names it as a stable body.

[0004] Stable bodies not only serve as a storage form of phosphorus, but also participate in a variety of biochemical reactions within cells, including DNA replication, repair, and protein synthesis. Stable bodies are also related to cell signal transduction. They can regulate the concentration of calcium ions within cells and affect cell proliferation and differentiation. In some cases, stable bodies may also participate in the cell's stress response, helping cells cope with changes in the external environment. In addition, researchers have found that stable bodies 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 vivo separation and extraction of stable bodies has become a hot topic in current biomedical research. However, this technology faces a series of challenges, including cell disruption, stable body release, separation, and purification. These steps require careful design to avoid degradation or alteration of the stable body structure and minimize interference from other components in the organism. Currently, the separation and extraction of stable bodies relies primarily on physical and chemical methods, including centrifugation, filtration, and chromatography. Although these methods have limitations in extraction efficiency, purity, and cost-effectiveness, they remain indispensable tools in current research. Guang et al. employed gel chromatography, which utilizes the size differences of stable body particles to achieve separation using a gel chromatography column. Although this method achieves good separation results, the required equipment is expensive, including high-performance liquid chromatography (HPLC) and sophisticated detectors. The high purchase cost of these equipment requires a significant investment in laboratories. Gel chromatography columns can fail due to the accumulation of impurities in the sample, requiring regular replacement to maintain separation efficiency, further increasing column costs. Furthermore, particulate matter and impurities in the sample can easily clog the column, affecting separation efficiency and reproducibility. Regular column maintenance and cleaning are required, increasing operational complexity and costs. Gel chromatography has high requirements for the 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-mentioned problems of gel chromatography limit its feasibility in large-scale applications. The density gradient centrifugation method proposed by Huang et al. uses a gradient formed by different density media (such as iodixanol and sucrose, etc.) to achieve the separation of 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. Moreover, during the density gradient centrifugation process, cells are easily broken, resulting in cytoplasmic contamination of the separated stable body particles, reducing the purity and activity of the stable body. In addition, the ultrafiltration method proposed in patent CN115678832 A uses ultrafiltration membranes of different pore sizes to achieve the separation and purification of exosome particles. It has the advantages of simple operation and suitability for large-scale separation, but it is difficult to separate the biological particles in the broken algae solution. Patent CN118028215 A proposes a method for extracting exosomes using ultracentrifugation. While this method can rapidly separate particles, the resulting exosomes are not highly purified and typically require combination with other separation methods to improve purity. While existing separation and extraction technologies each have their own advantages, further exploration and optimization of these methods, or the development of new technical strategies to overcome the limitations of existing technologies, are still needed to obtain high-purity, stable exosomes.

[0006] Natural stabilizers have applications in the medical, food, cosmetic, and agricultural sectors. In the medical sector, they can be used as components of drug delivery systems, leveraging their nanoscale size and high permeability to enhance drug distribution and absorption within the body; as vaccine adjuvants to boost immune responses and improve vaccine efficacy; and in regenerative medicine, as bioactive materials to promote cell proliferation and tissue repair. In the food sector, stabilizers can be used to develop functional foods with specific health benefits; as food additives to enhance nutritional value or improve food preservation properties. In the cosmetic sector, their bioactivity and nanoscale size allow them to be used as active ingredients to improve skin health and appearance; in oral care products, they can be used for antibacterial and anti-inflammatory effects, promoting oral tissue health. In agricultural applications, they can be used as biostimulants to promote crop growth and enhance disease and stress resistance; and as components of pesticides, their bioactivity can be used to control pests and pathogens. Consequently, stabilizers are in high demand in the market, but methods for extracting high-purity, contamination-free biosourced stabilizers have yet to be established. Summary of the Invention

[0007] The present invention aims to address the problem of low stabilization purity in existing stabilization extraction methods. The stabilization extracted using existing technologies contains a high level of impurities and byproducts, which not only affects its safety and effectiveness in clinical treatment but also limits its potential for application in scientific research. The present invention aims to provide a stabilization extraction method that can extract high-purity stabilization, has low health and environmental risks, and has broad adaptability. The extracted stabilization particles have high purity, good uniformity, and strong activity, meeting not only the application requirements of the food, daily chemical and other fields, but also the stringent requirements for application in 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 form from cyanobacteria, comprising the following steps:

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

[0011] S2. Cyanobacteria pretreatment: The cyanobacteria obtained in step S1 are washed, solid-liquid separated, and the cyanobacteria are collected;

[0012] S3. Ultrasonic disruption: The cyanobacteria obtained in step S2 were resuspended in a saline solution isotonic with the cyanobacteria and ultrasonically disrupted to obtain a cyanobacterial homogenate;

[0013] S4 differential centrifugation: The cyanobacterial homogenate obtained in step S3 was subjected to differential centrifugation to obtain a preliminary separation solution; large particles of impurities, including dead cells, EPS and other impurities, were removed by differential centrifugation in step S4 of the cyanobacterial homogenate obtained in step S3;

[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 are removed from the preliminary separation liquid obtained in step S4 by filtration 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, thereby obtaining a crude extract of the stable body, which contains a large amount of impurities (cell membranes, pigment particles, etc.).

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

[0016] S6 ultracentrifugation: The concentrate obtained in step S5 was resuspended in a NaCl aqueous solution or 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 resuspension; 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: Aqueous iodixanol solutions 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 the iodixanol suspension having a concentration of 10% w / v; density gradient centrifugation in step S7 was further performed to remove residual macromolecular non-stable particles and residual small-sized cell membranes in the crude stable body extract;

[0018] 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 resulting ultrafiltration concentrate is resuspended in a NaCl aqueous solution or buffer, ultrafiltered, and the concentrate is collected to obtain a high-purity stable body extract; step S8 eliminates residual cytoplasmic proteins in the crude stable body extract using Tween 20 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, an illumination intensity of 2000-4000lx, and a culture period of 14-28 days; the solid-liquid separation is performed by centrifugation; and 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 at 3000 to 8000 g for 10 to 20 min, preferably at 6000 g for 10 min; the washing and 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 the 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 minutes, preferably a frequency of 25 kHz and a time of 10 minutes.

[0022] In step S4, the differential centrifugation is performed under the following conditions: centrifuging the cyanobacteria homogenate obtained in step S3 at 300-600 g for 10-20 min, collecting the supernatant, centrifuging at 1000-3000 g for 10-20 min, collecting the supernatant, and centrifuging at 6000-12000 g for 20-40 min to collect the supernatant; preferably, the conditions are: centrifuging the cyanobacteria homogenate obtained in step S3 at 300 g for 10 min, collecting the supernatant, centrifuging at 2000 g for 10 min, collecting the supernatant, and centrifuging at 10000 g for 30 min to collect the supernatant.

[0023] In step S5, the filtration is water-based membrane filtration, preferably, filtration through 0.45 μm and 0.22 μm water-based membranes; and 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; and 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 centrifugation time of 1 to 2 h. Preferably, the temperature is 4° C., the centrifugal force is 150,000 g, and the centrifugation time is 2 h. In the ultracentrifuge tube, the amount of iodixanol aqueous solution of different concentrations added is the same; and the amount of the resuspension obtained in step S6 added is the same as the amount of iodixanol aqueous solution of each concentration added.

[0026] In step S8, the 10% w / v iodixanol suspension obtained in step S7 is mixed with 0.3% v / v Tween 20 in a volume ratio of 1:1-2; the room temperature oscillation is performed for 10-20 minutes, preferably 20 minutes; the ultrafiltration is performed, and the molecular weight cutoff of the ultrafiltration membrane is 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 produces a high-purity, stable extract. Furthermore, the process is stable and controllable, produces no toxic byproducts, and is environmentally friendly. It also does not require high-temperature, high-pressure equipment or large-scale instrumentation, expanding its application scenarios and reducing application costs. This method will address the technical challenges of preparing stable active granular biomaterials in China.

[0030] 2. The present invention can obtain high polyP n Polymerization, high concentration and high purity of stable active granular biomaterials, polyP n The degree of polymerization can reach 150 or above. The particle concentration of the stabilized extract obtained by the present invention is calculated to be 8.35×10 12 particles / L, and the granule protein concentration was 7.872×10 5 μg / L, and the particle-protein ratio was 1.061×10 7 The purity and concentration are very high, and the biological activity is high. It can not only meet the application requirements in the fields of food, daily chemicals, etc., 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 below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0032] Figure 1 This is a toluidine blue staining image of the stable body of 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 will be further described below with reference to the following examples. It should be understood that the following embodiments are intended to illustrate the present invention only and are not intended to limit the present invention. Specific techniques or conditions not specified in the examples were performed according to the techniques or conditions described in the literature in this area or according to the product instructions. Reagents or instruments used without manufacturer specified are conventional products available through regular channels.

[0037] In the following examples, the BG11 medium was purchased from Qingdao Hi-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 having mass concentrations of 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, and 2% w / v were prepared by diluting a 60% w / v iodixanol aqueous solution with pure water (OptiDensity Biotechnology Co., Ltd., Nanjing, China). TM Iodixanol solution, 60% w / v).

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

[0040] The biological source of the stable body in the following examples and comparative examples is hot spring cyanobacteria, which are separated and purified from algae samples collected in the wild. The specific methods are 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 group 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 liquid, use the streaking method to perform the initial coating on the culture medium to 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. Expansion 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 optimal survival temperature for 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 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 form from hot spring cyanobacteria, the extraction method comprising the following steps:

[0044] S1. Cultivation of Hot Spring Cyanobacteria: Cyanobacteria were inoculated into BG11 medium at a 10% v / v ratio and cultured at 50°C for 14 days with a 12h:12h light-dark ratio and a light intensity of 3000 lx. After the culture period, the algal suspension (300 mL) was collected and placed into six 50 mL centrifuge tubes. The cyanobacteria were enriched by centrifugation at 6000 g for 10 min, and the cyanobacterial pellet was collected.

[0045] S2. Cyanobacteria Pretreatment: Resuspend the cyanobacteria obtained in step S1 in 0.15 mM NaCl solution in six 50 mL centrifuge tubes. Centrifuge at 6000 g for 10 minutes in a high-speed centrifuge. Remove the supernatant. Repeat this wash four times, and finally collect the cyanobacteria pellet.

[0046] S3. Ultrasonic disruption: The cyanobacterial pellet 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 cyanobacterial homogenate;

[0047] S4. Differential Centrifugation: Separate the cyanobacterial homogenate obtained in Step 3 using a high-speed centrifuge. First, centrifuge at 300g for 10 minutes, remove the supernatant, then centrifuge at 2000g for 10 minutes, remove the supernatant again, and finally centrifuge at 10,000g for 30 minutes, remove the supernatant, and finally obtain six tubes of preliminary fractions.

[0048] S5. Ultrafiltration: The six tubes of preliminary separation liquid obtained in step S4 were first filtered through a 0.45 μm aqueous membrane, then filtered through a 0.22 μm aqueous membrane, and finally ultrafiltered using a 100 kDa ultrafiltration tube. The concentrate was collected, and 200 μL of concentrate was obtained from each tube. Finally, the concentrate was enriched together, totaling 1.2 mL.

[0049] S6. Ultracentrifugation: 1.2 mL of the 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. The precipitate was then resuspended in 2 mL of 45% w / v aqueous iodixanol to obtain a resuspension.

[0050] S7. Density gradient centrifugation: 2 mL of each 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. Then, 2 mL of the 45% w / v iodixanol resuspension obtained in step 6 was added to the ultracentrifuge tube. The tubes were ultracentrifuged at 150,000 g for 2 h at 4°C, and 2 mL of the 10% w / v iodixanol suspension was collected. Figure 3 This is a sample obtained after density gradient centrifugation in this example. 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. The red arrow in the figure points to the stable body particles.

[0051] S8. Removal of peripheral proteins: 3 mL of 0.3% (v / v) Tween 20 was added to 2 mL of 10% w / v iodixanol aqueous solution obtained in step S7, and the mixture was shaken at room temperature for 20 minutes. The Tween 20 was then removed by ultrafiltration through a 100 kDa ultrafiltration tube. The resulting concentrate was resuspended in 1 mL of 100 mM NaCl aqueous solution, ultrafiltered through a 100 kDa ultrafiltration tube, and the concentrate was collected. The resulting concentrate was again resuspended in 1 mL of 100 mM NaCl solution, and finally ultrafiltered through a 100 kDa ultrafiltration tube. The concentrate was collected to obtain 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, and the particle-protein ratio was 1.061×10 7 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 form from hot spring cyanobacteria, the extraction method comprising the following steps:

[0055] S1. Cultivation of Hot Spring Cyanobacteria: Cyanobacteria were inoculated into BG11 medium at a 10% v / v ratio and cultured at 50°C for 14 days with a 12h:12h light-dark ratio and a light intensity of 3000 lx. After the culture period, the algal suspension (300 mL) was collected and placed into six 50 mL centrifuge tubes. The cyanobacteria were enriched by centrifugation at 6000 g for 10 min, and the cyanobacterial pellet was collected.

[0056] S2. Cyanobacteria Pretreatment: Resuspend the cyanobacteria obtained in step S1 in 0.15 mM NaCl solution in six 50 mL centrifuge tubes. Centrifuge at 6000 g for 10 minutes in a high-speed centrifuge. Remove the supernatant. Repeat this wash four times, and finally collect the cyanobacteria pellet.

[0057] S3. Ultrasonic disruption: The cyanobacterial pellet 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 cyanobacterial homogenate;

[0058] S4. Differential Centrifugation: Separate the cyanobacterial homogenate obtained in Step 3 using a high-speed centrifuge. First, centrifuge at 300g for 10 minutes, remove the supernatant, then centrifuge at 2000g for 10 minutes, remove the supernatant again, and finally centrifuge at 10,000g for 30 minutes, remove the supernatant, and finally obtain six tubes of preliminary fractions.

[0059] S5. Ultrafiltration: The six tubes of preliminary separation liquid obtained in step S4 were first filtered through a 0.45 μm aqueous membrane, then filtered through a 0.22 μm aqueous membrane, and finally ultrafiltered using a 100 kDa ultrafiltration tube. The concentrate was collected, and 200 μL of concentrate was obtained from each tube. Finally, the concentrate was enriched together, totaling 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 granule protein concentration was 1.856×10 7 μg / L, and the granule protein ratio was 5.038×10 4 The concentration and purity of the stabilized extract are lower than those of the stabilized extract obtained in Example 1, and the stabilized extract can be applied to industries such as food, daily chemicals, and agriculture that do not require high purity of the stabilized extract.

[0061] Comparative Example 3

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

[0063] S1. Cultivation of Hot Spring Cyanobacteria: Cyanobacteria were inoculated into BG11 medium at a 10% v / v ratio and cultured at 50°C for 14 days with a 12h:12h light-dark ratio and a light intensity of 3000 lx. After the culture period, the algal suspension (300 mL) was collected and placed into six 50 mL centrifuge tubes. The cyanobacteria were enriched by centrifugation at 6000 g for 10 min, and the cyanobacterial pellet was collected.

[0064] S2. Cyanobacteria Pretreatment: Resuspend the cyanobacteria obtained in step S1 in 0.15 mM NaCl solution in six 50 mL centrifuge tubes. Centrifuge at 6000 g for 10 minutes in a high-speed centrifuge. Remove the supernatant. Repeat this wash four times, and finally collect the cyanobacteria pellet.

[0065] S3. Ultrasonic disruption: The cyanobacterial pellet 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 cyanobacterial homogenate;

[0066] S4. Differential Centrifugation: Separate the cyanobacterial homogenate obtained in Step 3 using a high-speed centrifuge. First, centrifuge at 300g for 10 minutes, remove the supernatant, then centrifuge at 2000g for 10 minutes, remove the supernatant again, and finally centrifuge at 10,000g for 30 minutes, remove the supernatant, and finally obtain six tubes of preliminary fractions.

[0067] S5. Ultrafiltration: The six tubes of preliminary separation liquid obtained in step S4 were first filtered through a 0.45 μm aqueous membrane, then filtered through a 0.22 μm aqueous membrane, and finally ultrafiltered using a 100 kDa ultrafiltration tube. The concentrate was collected, and 200 μL of concentrate was obtained from each tube. Finally, the concentrate was enriched together, totaling 1.2 mL.

[0068] S6. Ultracentrifugation: 1.2 mL of the 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. The precipitate was then resuspended in 2 mL of 45% w / v aqueous iodixanol to obtain a resuspension.

[0069] S7. Density gradient centrifugation: 2 mL of each 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. Then, 2 mL of the 45% w / v iodixanol resuspension obtained in step 6 was added to the ultracentrifuge tube. The tubes were ultracentrifuged at 150,000 g for 2 h at 4°C. 2 mL of the 10% w / v iodixanol suspension containing the stable 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 1 and Comparative Example 3, the cytoplasmic protein in the stable body extract was not removed by TWeen20 in Comparative Example 3, and the stable body granule protein ratio obtained was 1.534×10 6 particles / μg, while in Example 1, the cytoplasmic proteins in the stable extract were 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] This 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, and meets 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 applicable to different PolyP n The polymericity and bio-derived stability of this invention have driven innovative research in fields such as biomedicine, materials science, and environmental science. Furthermore, this invention supports the development of clinical treatment plans, improves patients' quality of life, and promotes the development of related industries, including biopharmaceuticals, health products, and environmental governance, opening up new opportunities and breakthroughs for clinical treatment and scientific research.

[0072] The present invention provides a concept and method for extracting a stabilized form from cyanobacteria. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A method for extracting a stable form from cyanobacteria, characterized in that: The steps include: S1. Cyanobacteria cultivation: Cyanobacteria are inoculated into a blue-green algae culture medium, cultured, solid-liquid separated, and the cyanobacteria are collected; S2. Cyanobacteria pretreatment: The cyanobacteria obtained in step S1 are washed, solid-liquid separated, and the cyanobacteria are collected; S3 ultrasonic disruption: The cyanobacteria obtained in step S2 were resuspended in a saline solution isotonic with the cyanobacteria and ultrasonically disrupted to obtain a cyanobacterial homogenate; the ultrasound having a frequency of 20 to 25 kHz and an ultrasonic time of 5 to 10 min; S4. Differential centrifugation: The cyanobacterial homogenate obtained in step S3 was subjected to differential centrifugation to obtain a preliminary separated liquid; the differential centrifugation conditions are as follows: the cyanobacterial homogenate obtained in step S3 was centrifuged at 300-600g for 10-20min, the supernatant was collected, the supernatant was collected by centrifugation at 1000-3000g for 10-20min, the supernatant was collected, and the supernatant was collected by centrifugation at 6000-12000g for 20-40min. S5. Ultrafiltration: The preliminary separated liquid obtained in step S4 was filtered, ultrafiltered, and the concentrate was collected to obtain a stable crude extract; the filtration was performed through a 0.45 μm and 0.22 μm water filter membrane; the ultrafiltration, the molecular weight cutoff of the ultrafiltration membrane was 100 kDa; 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 resuspension; the ultracentrifugation temperature was 4 to 10 ° C, the centrifugal force was 120,000 to 150,000 g, and the centrifugation time was 1 to 2 h; 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; the ultracentrifugation was performed at a temperature of 4 to 10 ° C, a centrifugal force of 120,000 to 150,000 g, and a centrifugation time of 1 to 2 h; 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 buffer, ultrafiltered, and the concentrate is collected to obtain a high-purity stable body extract.

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

3. The method according to claim 1, characterized in that In step S2, the washing is performed using a NaCl aqueous solution or a PBS buffer solution; the solid-liquid separation is performed by centrifugation; the centrifugation is performed at 3000 to 8000 g for 10 to 20 minutes; the washing and 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 the centrifuge tube.

4. The method according to claim 3, characterized in that In step S2, 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.

5. The method according to claim 1, characterized in that In step S3, the salt solution isotonic with the cyanobacteria is a NaCl aqueous solution.

6. The method according to claim 5, characterized in that In step S3, the salt solution isotonic with the cyanobacteria is preferably a NaCl aqueous solution with a concentration of 0.15 mM.

7. The method according to claim 1, characterized in that In step S6, the NaCl aqueous solution is a 0.15 mM NaCl aqueous solution; the buffer solution is a PBS buffer solution with a concentration of 5 to 10 mM and a pH of 7.2 to 7.4; and the concentration of the iodixanol aqueous solution is 45 to 55% w / v.

8. The method according to claim 1, characterized in that In step S7, the same amount of iodixanol aqueous solutions of different concentrations are added to the ultracentrifuge tube; and the same amount of the resuspension obtained in step S6 is added as the amount of the iodixanol aqueous solutions of each concentration.

9. The method according to claim 1, characterized in that In step S8, the 10% w / v iodixanol suspension obtained in step S7 is mixed with 0.3% v / v Tween 20 in a volume ratio of 1:1-2; the room temperature oscillation is performed for 10-20 minutes; the ultrafiltration is performed, and the molecular weight cutoff of the ultrafiltration membrane is 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 the buffer, ultrafiltration, and collecting the concentrate are repeated 1-10 times.

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