A method for preparing functional polypeptides from Spirulina

Abstract

This invention discloses a method for preparing a functional polypeptide from Spirulina. The polypeptide provided by this invention has the amino acid sequence shown in Sequence 1 of the sequence listing. The functional polypeptide (YSDITRPG) discovered in this invention from Spirulina is a pure, natural, non-toxic, and harmless microbial-derived substance, and possesses significant ACE inhibitory and colon cancer cell inhibitory effects. It can be used as a blood pressure lowering component or as an active ingredient in colon cancer drugs. It is non-toxic, meets the requirements for pharmaceutical formulation development, and has good market prospects in the pharmaceutical industry.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance extraction technology, specifically to a method for preparing functional polypeptides from spirulina. Background Technology

[0002] Spirulina, a microalga with high nutritional value, contains 60%-70% high-quality protein and is typically processed into spirulina peptides. However, traditional peptide extraction methods (such as organic solvent extraction, acid / alkali hydrolysis, or enzymatic hydrolysis) generally suffer from problems such as high solvent toxicity and loss of peptide activity under high temperature or extreme pH conditions. Furthermore, subsequent purification steps are cumbersome, limiting its industrial application. In addition, the organic wastewater generated by traditional processes places a significant burden on the environment, making it difficult to meet the needs of green and sustainable development.

[0003] In recent years, deep eutectic solvents have attracted widespread attention in the field of natural product extraction due to their designability, low toxicity, high biocompatibility, and excellent solubility. Deep eutectic solvents are typically composed of hydrogen bond donors and acceptors, and their physicochemical properties can be precisely controlled by adjusting the component ratios.

[0004] In existing technologies, peptide extraction and purification processes are often separated, resulting in lengthy processes and significant loss of active ingredients. There is an urgent need to develop an integrated extraction and purification technology that is efficient, environmentally friendly, and maximizes the preservation of peptide bioactivity. Furthermore, how to achieve efficient disruption of phycocyanin cell walls and selective dissolution of target peptides through the rational design of deep eutectic solvents remains a critical technical challenge to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing functional peptides from Spirulina. The deep eutectic solvent used can dissolve the crude Spirulina peptide extract under mild conditions while maintaining its bioactivity. Compared with traditional extraction processes, the deep eutectic solvent used in this invention has the characteristics of biodegradability and low cytotoxicity. Furthermore, target components were screened from the crude Spirulina peptide extract, and their structures were further identified, confirming their amino acid sequences. The Spirulina peptides of this invention exhibit significant antioxidant, antibacterial, angiotensin-transferase inhibitory, and colon cancer cell inhibitory activities, showing broad application prospects in the pharmaceutical and cosmetic fields.

[0006] The purpose of this application is not limited to the above-mentioned purposes. Other purposes and advantages of this application not mentioned above can be understood from the following description and will become clearer through the embodiments of this application. Furthermore, it is readily understood that the purposes and advantages of this application can be achieved through the features disclosed in the claims and combinations thereof.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention protects a method for extracting spirulina polypeptides, comprising the following steps: Spirulina polypeptide source material is mixed with a deep eutectic solvent for extraction to obtain crude spirulina polypeptide extract; wherein, the deep eutectic solvent includes betaine, polyol and water in a molar ratio of 1:(2-3):(0-1), wherein the polyol is glycerol or 1,3-propanediol.

[0008] According to an embodiment of the present invention, preferably, the eutectic solvent is betaine and 1,3-propanediol in a molar ratio of 1:3.

[0009] According to an embodiment of the present invention, preferably, the spirulina polypeptide source material includes phycocyanin extracted from Spirulina platensis powder.

[0010] According to an embodiment of the present invention, preferably, the solid-liquid ratio of the spirulina polypeptide source material and the deep eutectic solvent is 1: (40-120) g / mL; the extraction is carried out under shaking conditions, at a temperature of 65-85°C, at a speed of 800-1200 rpm, and for a time of 6-30 h.

[0011] According to an embodiment of the present invention, the method further includes the following step: separating and collecting components with a molecular weight >500 Da from the spirulina polypeptide extract obtained in the extraction step.

[0012] Secondly, the present invention provides a crude extract of spirulina polypeptide obtained by any of the methods described above.

[0013] Thirdly, the present invention provides the use of the crude spirulina polypeptide extract in any of the following (a1)-(a4): (a1) In vitro antioxidant activity not for therapeutic purposes; (a2) Preparation of antioxidant products; (a3) In vitro inhibition of Staphylococcus aureus and / or Escherichia coli for non-therapeutic purposes; (a4) Prepare disinfectants or drugs that inhibit Staphylococcus aureus and / or Escherichia coli.

[0014] Fourthly, the present invention provides a method for preparing spirulina polypeptides, comprising the step of extracting crude spirulina polypeptides using any of the methods described above, and the following steps: The crude extract of Spirulina polypeptide was dissolved in water to prepare a polypeptide solution of 200 mg / mL, which was then loaded into an AKTA chromatography system for elution. The AKTA chromatography system used a Superdex 75 pg column, a flow rate of 2 mL / min, and a column pressure of 0.50 MPa. 10 mL of eluent was used as one tube, for a total of 36 tubes. The fraction from the 23rd tube was collected.

[0015] Fifthly, the present invention provides spirulina polypeptides extracted by the method described above.

[0016] Sixthly, the present invention provides the use of the spirulina polypeptide in any of the following (b1)-(b9): (b1) In vitro antioxidant activity for non-therapeutic purposes; (b2) Preparation of antioxidant products; (b3) In vitro inhibition of Staphylococcus aureus and / or Escherichia coli for non-therapeutic purposes; (b4) Prepare disinfectants or drugs that inhibit Staphylococcus aureus and / or Escherichia coli; (b5) Preparation of angiotensin-transferase inhibitors; (b6) To prepare drugs for the prevention and / or treatment of hypertension; (b8) In vitro inhibition of colon cancer cells for non-therapeutic purposes; (b9) Prepare drugs for the prevention and / or treatment of colon cancer.

[0017] In a seventh aspect, the present invention provides a polypeptide whose amino acid sequence is shown in Sequence 1 of the sequence listing.

[0018] Eighthly, the present invention provides the use of the polypeptide shown in sequence 1 in any of the following (1c)-(c9): (c1) In vitro antioxidant activity for non-therapeutic purposes; (c2) Preparation of antioxidant products; (c3) In vitro inhibition of Staphylococcus aureus and / or Escherichia coli for non-therapeutic purposes; (c4) Prepare disinfectants or drugs that inhibit Staphylococcus aureus and / or Escherichia coli; (c5) Preparation of angiotensin-transferase inhibitors; (c6) Prepare drugs for the prevention and / or treatment of hypertension; (c8) In vitro inhibition of colon cancer cells for non-therapeutic purposes; (c9) Prepare drugs for the prevention and / or treatment of colon cancer.

[0019] In a ninth aspect, the present invention provides a product of any one of (d1)-(d5) below, wherein the active ingredient is the polypeptide shown in sequence 1; (d1) Antioxidant drugs or antioxidant cosmetics; (d2) Disinfectants or drugs that inhibit Staphylococcus aureus and / or Escherichia coli; (d3) Angiotensin-transferase inhibitors; (d5) Medications for treating colon cancer.

[0020] The present invention has the following beneficial effects: (1) This invention develops a green extraction technology based on deep eutectic solvent. The DES system of this invention can significantly improve the extraction efficiency of Spirulina peptides while avoiding the environmental pollution problems of traditional organic solvents. At the same time, this invention screens the optimal DES combination based on peptide yield and activity evaluation: betaine and 1,3-propanediol in a molar ratio of 1:3.

[0021] (2) In this invention, the target polypeptide is first preliminarily enriched from the crude extract by dialysis technology, and then finely separated by molecular size exclusion chromatography (AKTA purification system) to finally obtain five Spirulina polypeptide components (SP-1 to SP-5) with similar molecular weight distribution. The component with the best activity was screened by in vitro antioxidant activity (such as DPPH) and antibacterial experiment (inhibitory effect on Staphylococcus aureus and Escherichia coli). The activity and structure of the component were then tested and identified, laying the foundation for elucidating its structure-activity relationship.

[0022] This invention establishes a set of efficient and green peptide separation and purification processes, providing a new strategy for the green extraction and high-value utilization of spirulina peptides, which has important scientific significance and application value.

[0023] (3) The Spirulina polypeptide extract obtained by the present invention has significant antioxidant, antibacterial, angiotensin-converting enzyme inhibition, colon cancer cell inhibition and other physiological activities, and has broad application prospects in the fields of medicine and cosmetics.

[0024] (4) The functional polypeptide (YSDITRPG) discovered in Spirulina in this invention is a pure, natural, non-toxic, and harmless microbial-derived substance, and it has significant ACE inhibitory effects and colorectal cancer cell inhibitory effects. As a blood pressure lowering component, YSDITRPG of this invention has a good ACE inhibitory effect and can also be used as an active ingredient in colorectal cancer drugs. It is non-toxic, meets the requirements of drug formulation development, and has good market prospects in the pharmaceutical industry. Attached Figure Description

[0025] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The figure shows the extraction yield of Spirulina from different deep eutectic solvents of betaine-polyols.

[0026] Figure 2 The results of antibacterial experiments on Staphylococcus aureus (left) and Escherichia coli (right) by crude extracts of Spirulina polypeptides extracted with different deep eutectic solvents of betaine-polyols.

[0027] Figure 3 The chromatogram of Spirulina polypeptides purified by AKTA protein chromatography.

[0028] Figure 4 The nonlinear fitting plot of IC50 for SP-4 DPPH clearance rate.

[0029] Figure 5 The results of antibacterial experiments on Staphylococcus aureus (left) and Escherichia coli (right) by SP-1, SP-2, SP-3, SP-4 and SP-5.

[0030] Figure 6 This is a statistical chart showing the diameter of the inhibition zone of SP-4 against Staphylococcus aureus and Escherichia coli at different concentrations.

[0031] Figure 7 The graph shows the linear fit between SP-4 concentration and angiotensin-converting enzyme inhibition rate.

[0032] Figure 8 This is a graph showing the cell viability analysis after incubation of SP-4 at different concentrations for 24 h and 48 h.

[0033] Figure 9 To examine the phase map of the total cell cycle at different concentrations of SP-4 using flow cytometry.

[0034] Figure 10 This is the gel permeation chromatography spectrum of SP-4.

[0035] Figure 11 This is a table showing the results of the SP-4 amino acid analyzer.

[0036] Figure 12 The amino acid sequence spectrum of SP-4 was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (A - primary spectrum, B - secondary spectrum). Detailed Implementation

[0037] The present application will now be described in further detail with reference to the embodiments. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit the invention.

[0038] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0039] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0040] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0041] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0042] As mentioned earlier, existing traditional peptide extraction methods for spirulina peptides generally suffer from problems such as high solvent toxicity, loss of peptide activity due to high temperatures or extreme pH conditions, and cumbersome subsequent purification steps. In view of this, the inventors of this application have conducted extensive research on solvents that can be used as spirulina peptide extractants and their extraction technologies, and have developed a green extraction technology based on deep eutectic solvents.

[0043] In the first part, the present invention provides a method for extracting spirulina polypeptides, comprising the following steps: mixing spirulina polypeptide source material with a deep eutectic solvent for extraction to obtain crude spirulina polypeptide extract; wherein, the deep eutectic solvent comprises betaine, polyol and water in a molar ratio of 1:(2-3):(0-1), wherein the polyol is glycerol or 1,3-propanediol.

[0044] The term "spirulina" as used in this application refers to an economically important microalga belonging to the family Oscillatidae in the phylum Cyanobacteria, which is classified as Cyanobacteria.

[0045] The term "eutectic solvent" as used in this application, also known as a low-eutectic solvent, is a eutectic mixture formed by hydrogen bonding between two or more components, which is liquid at room temperature. Its components include hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs). Low-eutectic solvents can be formed by any method known in the art. For example, compounds that are solid at 25°C and are in a solid state (e.g., in powder form) can be directly mixed, then slightly heated to melt and completely homogenize the solid, followed by cooling. The resulting low-eutectic solvent remains liquid at 25°C. Additional liquid components (e.g., water or glycerol) can be added to the aforementioned compounds to promote the formation of low-eutectic solvents.

[0046] According to embodiments of the present invention, a component may act as a hydrogen bond acceptor in one specific combination and as a hydrogen bond donor in another specific combination, or may act as a hydrogen bond acceptor and donor for each other with another component in one specific combination. Exemplarily, when the eutectic solvent comprises betaine-glycerol-water, betaine is the hydrogen bond acceptor and glycerol is the hydrogen bond donor; when it comprises betaine-1,3-propanediol, betaine is the hydrogen bond acceptor and 1,3-propanediol is the hydrogen bond donor; when it comprises betaine-ethylene glycol, betaine is the hydrogen bond acceptor and ethylene glycol is the hydrogen bond donor.

[0047] This application research has discovered that mixing betaine, polyol (and water) in a certain molar ratio can form a deep eutectic solvent for the extraction of spirulina polypeptides. This deep eutectic solvent has the advantages of being non-toxic, having low operating costs, good selectivity, easy control of reaction conditions, and being recyclable. This application is based on the above findings.

[0048] The applicant designed three different DES systems (betaine-glycerol-water, betaine-1,3-propanediol, and betaine-ethylene glycol), systematically optimized key process parameters such as solid-liquid ratio, extraction temperature, and extraction time, and screened the optimal DES combination based on peptide yield and activity evaluation. Experimental results show that this DES system can significantly improve the extraction efficiency of spirulina peptides while avoiding the environmental pollution problems of traditional organic solvents. The inventors unexpectedly discovered that, compared to betaine-ethylene glycol, using a deep eutectic solvent of betaine, polyol (glycerol or 1,3-propanediol), and water in a molar ratio of 1:(2-3):(0-1) to extract spirulina peptides not only resulted in a higher extraction rate but also higher antioxidant and antibacterial activity in the extracted spirulina peptides.

[0049] This application uses antioxidant activity and antibacterial activity as detection indicators to screen DES systems. Further, in some preferred embodiments of this application, the spirulina peptides extracted by the DES are mixed with a DPPH sample solution, and the DPPH free radical scavenging rate is measured to evaluate the antioxidant capacity. Optionally, the concentration of the DPPH reagent is 2 × 10⁻⁶. -4 The concentration of spirulina polypeptide extracted by DES was 5 mg / mL, the reaction time was 30 min, the reaction conditions were light-protected at room temperature, and the detection wavelength was 517 nm. Further, in some preferred embodiments of this application, the antibacterial effect of the DES-extracted spirulina polypeptide against Staphylococcus aureus and Escherichia coli was determined using a perforation method to evaluate its antibacterial ability. Optionally, the concentration of the bacterial suspension was 1 × 10⁻⁶. 6 -10 7 The concentration of CFU / mL was 0.85% sterile sodium chloride solution for bacterial suspension preparation. The concentration of spirulina polypeptide extracted by DES was 150 mg / mL. The culture conditions for Staphylococcus aureus and Escherichia coli were 37℃ for 12 h.

[0050] According to an embodiment of the present invention, more preferably, the eutectic solvent is a molar ratio of betaine and 1,3-propanediol of 1:3. The inventors have found that the extraction rate, antioxidant activity, and antibacterial activity of the spirulina polypeptides are all highest when using this eutectic solvent.

[0051] Based on the above technical solutions, the deep eutectic solvents used in this invention are all selected from natural ingredients and can be used as additives in cosmetics, etc. The prepared natural deep eutectic solvents are not only non-toxic but also chemically stable, achieving better extraction results with less damage to the structure of spirulina polypeptides. They can replace traditional organic solvents as a novel green extraction agent for spirulina polypeptides. The extraction method using this solvent is safer, can maximize the preservation of spirulina polypeptide activity, and alleviates the drawbacks of traditional extraction methods, such as cumbersome processes and environmental unfriendliness. The deep eutectic solvent is liquid at room temperature. Room temperature refers to a temperature of 20-35°C. Further, in some preferred embodiments of this application, the preparation method of the deep eutectic solvent is as follows: the raw materials are mixed evenly at 70-90°C according to a predetermined amount to form the liquid deep eutectic solvent.

[0052] According to embodiments of the present invention, the spirulina polypeptide source material is a material rich in spirulina polypeptides, such as spirulina polypeptides extracted from Spirulina platensis and Spirulina macrophylla, including but not limited to phycocyanin extracted from Spirulina platensis powder. Optionally, protein extraction and purification are performed using an ultrasonic disruptor, repeated freeze-thaw cycles for cell disruption, and stepwise salting out.

[0053] The term "cell disruption" as used in this application refers to the process of breaking the integrity of spirulina cells, specifically the disruption of the cell wall of spirulina, which allows the release of its internal components, such as the release of phycocyanin stored in the cells.

[0054] Furthermore, in some preferred embodiments of this application, ultrasonic disruption and repeated freeze-thaw cycles are used for cell disruption, and ammonium sulfate is used for stepwise salting out of the protein for purification. Optionally, the mass ratio of the Spirulina platensis powder to deionized water is 1:50. The ultrasonic disruption conditions are: power 700 W, ultrasonic treatment for 10 seconds followed by a 10-second interval, for a total treatment time of 30 minutes. The repeated freeze-thaw conditions are: freezing in liquid nitrogen at 40°C, thawing at room temperature, and repeating the freeze-thaw cycle 4 times. The salting out purification conditions are: stepwise salting out (30% to 50% saturation). Optimizing the repeated freeze-thaw treatment method, the number of freeze-thaw cycles, and the stepwise salting out saturation allows for the full dissolution of phycocyanin, achieving a better extraction effect.

[0055] Furthermore, in some preferred embodiments of this application, the precipitate was collected and dissolved in 0.05 M PBS buffer (pH 6.86). The dissolved solution was placed in a dialysis bag and dialyzed at 4 °C for desalination. The dialysis endpoint was determined by titration with 0.2% barium chloride solution. After dialysis, the solution was freeze-dried under vacuum to obtain phycocyanin.

[0056] This application does not impose any special limitations on the mixing process; any mixing process known to those skilled in the art can be used.

[0057] According to embodiments of the present invention, the solid-liquid ratio of the spirulina polypeptide source material to the deep eutectic solvent is 1:(40-120) g / mL; typical but non-limiting solid-liquid ratios of phycocyanin to the deep eutectic solvent are, for example, 1:40 g / ml (w / v), 1:60 g / ml (w / v), 1:80 g / ml (w / v), 1:100 g / ml (w / v), and 1:120 g / ml (w / v). Optimizing the ratio of the spirulina polypeptide source material to the deep eutectic solvent can achieve better extraction efficiency, more thorough extraction, and save on the cost of the raw materials.

[0058] According to embodiments of the present invention, the extraction is carried out under oscillation conditions at a temperature of 65–85°C, a speed of 800–1200 rpm, and a time of 6–30 h. Typical but non-limiting oscillation speeds are, for example, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm, 1200 rpm, or any value between two adjacent values. Typical but non-limiting processing temperatures are 65°C, 70°C, 75°C, 80°C, and 85°C; typical but non-limiting oscillation times are 6 h, 12 h, 18 h, 24 h, and 30 h. By optimizing the solid-liquid ratio, extraction temperature, and time, the spirulina polypeptides in the spirulina polypeptide source material can be fully separated from the material and enter the deep eutectic solvent, achieving better extraction and separation effects.

[0059] Furthermore, in some preferred embodiments of this application, solid-liquid separation is performed after shaking treatment to obtain spirulina polypeptide extract.

[0060] This application does not impose any special limitations on solid-liquid separation; the separation method can be selected from any one of filtration, centrifugation, gravity sedimentation, or centrifugal sedimentation, with centrifugation being preferred. The centrifugation speed is preferably 5000–10000 rpm, more preferably 8000 rpm, and the centrifugation time is preferably 15–25 min, more preferably 20 min.

[0061] Furthermore, in some preferred embodiments of this application, the method further includes the following step: separating and collecting components with a molecular weight >500 Da from the spirulina polypeptide extract obtained in the extraction step. After numerous attempts, the inventors ultimately chose to separate and collect components >500 Da to obtain crude polypeptide extracts and polypeptides with the desired properties.

[0062] This application does not impose any particular limitation on the method for separating and collecting components with the target molecular weight; it may be selected from any one of dialysis, ultrafiltration, density gradient centrifugation, or gel size exclusion chromatography, with dialysis being preferred. Optionally, the spirulina polypeptide extract is placed in a dialysis bag with a molecular weight cutoff of 500 Da, the dialysis bag is placed in ultrapure water, and dialysis is performed at 4°C to retain spirulina polypeptides with a molecular weight cutoff > 500 Da. After drying, spirulina polypeptides are obtained.

[0063] This application does not impose any special restrictions on the drying method, which can be selected from spray drying or freeze drying.

[0064] In the second part, this invention provides a crude extract of spirulina polypeptides obtained by any of the methods described above. Compared to the combination of betaine and ethylene glycol, a deep eutectic solvent composed of betaine, polyol (glycerol or 1,3-propanediol), and water in a molar ratio of 1:(2-3):(0-1) yields a higher extraction rate of spirulina polypeptides and exhibits higher antioxidant and antibacterial activity, especially a deep eutectic solvent composed of betaine and 1,3-propanediol in a molar ratio of 1:3.

[0065] Part Three, the present invention provides the use of the crude spirulina polypeptide extract in any of the following (a1)-(a4): (a1) In vitro antioxidant activity not for therapeutic purposes; (a2) Preparation of antioxidant products; (a3) In vitro inhibition of Staphylococcus aureus and / or Escherichia coli for non-therapeutic purposes; (a4) Prepare disinfectants or drugs that inhibit Staphylococcus aureus and / or Escherichia coli.

[0066] According to embodiments of the present invention, this application does not impose any special limitations on the in vitro antioxidant for non-therapeutic purposes. Exemplarily, the in vitro antioxidant for non-therapeutic purposes may be a skin care product using the spirulina polypeptide or a skin care product with the spirulina polypeptide as an active ingredient.

[0067] According to embodiments of the present invention, this application does not specifically limit the in vitro antioxidant for non-therapeutic purposes. Exemplarily, the antioxidant product may be an antioxidant drug. The antioxidant product can help prevent or delay disease progression by scavenging excess free radicals.

[0068] According to embodiments of the present invention, this application does not specifically limit the in vitro inhibition of Staphylococcus aureus and / or Escherichia coli for non-therapeutic purposes. Exemplarily, the in vitro inhibition of Staphylococcus aureus and / or Escherichia coli for non-therapeutic purposes may be the inhibition of Staphylococcus aureus and / or Escherichia coli on the skin surface (non-broken skin surface with an intact barrier) of the human or other animal using the spirulina polypeptide or a product with the spirulina polypeptide as an active ingredient, or the inhibition of Staphylococcus aureus and / or Escherichia coli in the external environment.

[0069] According to embodiments of the present invention, this application does not specifically limit the product that inhibits Staphylococcus aureus and / or Escherichia coli. Exemplarily, the product that inhibits Staphylococcus aureus and / or Escherichia coli may be a drug or disinfectant.

[0070] Part Four, this invention provides a method for preparing spirulina polypeptides, comprising extracting crude spirulina polypeptides using the method described in any one of the preceding descriptions and the following steps: The crude extract of Spirulina polypeptide was dissolved in water to prepare a polypeptide solution of 200 mg / mL, which was then loaded into an AKTA chromatography system for elution. The AKTA chromatography system used a Superdex 75 pg column, a flow rate of 2 mL / min, and a column pressure of 0.50 MPa. 10 mL of eluent was used as one tube, for a total of 36 tubes. The fraction from the 23rd tube was collected.

[0071] Based on the above technical solutions, this application establishes a highly efficient and green peptide separation and purification process: First, the target peptide is initially enriched using dialysis technology, and then finely separated using size exclusion chromatography (AKTA purification system), ultimately obtaining five Spirulina peptide fractions (SP-1 to SP-5) with similar molecular weight distributions. The fraction with the best activity is screened through in vitro antioxidant activity (such as DPPH) and antibacterial experiments (inhibitory effects against Staphylococcus aureus and Escherichia coli).

[0072] The term "AKTA chromatography system" as used in this application refers to a modular liquid chromatography purification system consisting of a pump, detector, autosampler, fraction collector, and control software, specifically designed for the separation and purification of biological macromolecules (such as proteins and peptides).

[0073] Furthermore, in some preferred embodiments of this application, to meet the experimental requirements for separating and purifying spirulina peptides using the AKTA system, the prepared buffer solution is passed through a Superdex™ 75 pg chromatography column at a column pre-pressure of 0.50 MPa and a constant flow rate of 2 mL / min, and continuously washed for 4 h to ensure that the chromatography column is fully equilibrated and to remove residual impurities within the column. Exemplarily, the buffer solution is a pH 8.0 Tris-NaCl buffer (20 mM Tris, 100 mM NaCl), and the preparation steps specifically include: weighing 12.114 g of Tris and dissolving it in approximately 1.6 L of ultrapure water, stirring thoroughly until completely dissolved; then adding 11.688 g of NaCl, continuing to stir until completely dissolved; monitoring the pH value of the solution using a pH meter, and adjusting the pH to 8.0 by adding HCl solution dropwise; finally, bringing the solution to a final volume of 2 L, mixing thoroughly, and storing for later use. Optionally, after the buffer solution is prepared, it is subjected to ultrafiltration using a 0.22 μm filter membrane to remove impurities and microorganisms; after ultrafiltration, the buffer solution is placed in an ultrasonic cleaner for 30 minutes of ultrasonic treatment to remove residual air bubbles and ensure the homogeneity and stability of the buffer solution.

[0074] Furthermore, in some preferred embodiments of this application, during the elution process, the signal change of the elution peak is monitored in real time at a wavelength of 280 nm using an ultraviolet detector, and the eluent corresponding to the target peak is collected; after elution, the collected fractions are freeze-dried and stored at 4°C in the dark for later use.

[0075] Part Five, this invention provides the spirulina polypeptide described in any of the preceding claims. The applicant screened SP-4 by conducting antioxidant and antibacterial experiments on different components, including those after freeze-drying.

[0076] Optionally, the antioxidant experiment uses DPPH scavenging rate as an indicator, and the DPPH stock solution concentration is 2×10⁻⁶. -3 mol / L, DPPH working solution concentration is 2×10 -4 The sample solution concentration was 5 mg / mL (diluted with ethanol). The sample treatment conditions were: the sample solution and DPPH working solution were mixed 1:1, the reaction conditions were: protected from light for 30 min, and the measurement wavelength was 517 nm.

[0077] Optionally, the antibacterial experiment uses the filter paper diffusion method to study the antibacterial effect. BHI and LB liquid media are used to prepare bacterial suspensions of Staphylococcus aureus and Escherichia coli, with a suspension concentration of 1×10⁻⁶. 6 -10 7 The concentration of CFU / mL was 75 mg / mL for different component sample solutions, and the culture conditions were 37℃ for 12 h.

[0078] The applicant further conducted activity tests on the target substance SP-4.

[0079] Furthermore, in some preferred embodiments of this application, the antioxidant activity of SP-4 was determined using a DPPH free radical scavenging assay.

[0080] Optionally, the reaction conditions are: DPPH stock solution concentration of 2 × 10⁻⁶. -3 mol / L, DPPH working solution concentration is 2×10 -4 Sample solutions were prepared at concentrations of 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, and 8 mg / mL (diluted with ethanol). Sample treatment conditions included mixing the sample solution with DPPH working solution at a 1:1 ratio, reacting in the dark for 30 min, and measuring at a wavelength of 517 nm. The results showed that the IC50 of SP-4 was [missing information - likely a specific concentration]. 50 The value was 3.89 ± 0.45 mg / mL.

[0081] Furthermore, in some preferred embodiments of this application, the antibacterial effect of SP-4 against Staphylococcus aureus and Escherichia coli was determined using a perforation method.

[0082] Optionally, the experimental method is as follows: SP-4 sample solutions with concentrations of 200 mg / mL, 180 mg / mL, 170 mg / mL, 150 mg / mL, 125 mg / mL, 100 mg / mL, 70 mg / mL, and 50 mg / mL are prepared as bacterial suspensions using a 0.85% sodium chloride solution, with a bacterial suspension concentration of 1×10⁻⁶. 6 -10 7 The concentration of the different component samples was 75 mg / mL, and the culture conditions were 37℃ for 12 h. The results showed that at a concentration of 200 mg / mL, the inhibition zone diameter against Staphylococcus aureus reached its maximum value (1.3±0.06 cm), and the inhibition zone diameter against Escherichia coli reached its maximum value (2±0.1 cm), which was significantly higher than that of the low concentration group.

[0083] Part VI, the present invention provides the use of the spirulina polypeptide described above in any of the following (b1)-(b9): (b1) In vitro antioxidant activity for non-therapeutic purposes; (b2) Preparation of antioxidant products; (b3) In vitro inhibition of Staphylococcus aureus and / or Escherichia coli for non-therapeutic purposes; (b4) Prepare products that inhibit Staphylococcus aureus and / or Escherichia coli; (b5) Preparation of angiotensin-transferase inhibitors; (b6) To prepare drugs for the prevention and / or treatment of hypertension; (b8) In vitro inhibition of colon cancer cells for non-therapeutic purposes; (b9) Prepare drugs for the prevention and / or treatment of colon cancer.

[0084] According to an embodiment of the present invention, the relevant explanations of (b1)-(b4) are the same as above.

[0085] Further, in some preferred embodiments of this application, high-performance liquid chromatography (HPLC) is used to detect the content of hippuric acid generated in the reaction and to determine the angiotensin-converting enzyme (ACE) inhibitory activity. Specifically, this includes: preparing a 6.5 mM HHL standard solution and a 0.1 U / mL ACE solution, preparing the reaction system, reacting at 37°C for 30 min, and then adding acetonitrile to terminate the reaction. Optionally, the SP-4 preparation solution is a borate-borax buffer solution, the SP-4 sample concentration is 2 mg / mL, and the HPLC analysis conditions are: Hypersil ODS2 column (200 mm × 4.6 mm, 5 μm), mobile phase of 25% acetonitrile and 75% ultrapure water (containing 0.1% TFA), flow rate of 1 mL / min; detection wavelength of 228 nm, column temperature of 30°C, and injection volume of 20 μL.

[0086] The term "angiotensin-converting enzyme inhibitor" as used in this application refers to a substance capable of inhibiting the activity of angiotensin-converting enzyme. "Angiotensin-converting enzyme inhibitory activity" is the efficiency by which a sample inhibits the catalytic formation of hippuric acid by angiotensin-converting enzyme, and the inhibition rate is calculated using the peak area at a wavelength of 228 nm, as determined by high-performance liquid chromatography.

[0087] Angiotensin-converting enzyme (ACE) plays an important role in catalyzing the conversion of angiotensin I to angiotensin II and regulating bradykinin. It is often studied extensively as a target for controlling blood pressure levels. The spirulina polypeptide of this invention can achieve the effect of lowering blood pressure by inhibiting the activity of ACE.

[0088] According to embodiments of the present invention, this application does not have any particular limitation on the in vitro inhibition of colon cancer cells for non-therapeutic purposes. Exemplarily, the in vitro inhibition of colon cancer cells for non-therapeutic purposes may be a screening of related drugs using the spirulina polypeptide or a product with the spirulina polypeptide as an active ingredient.

[0089] According to embodiments of the present invention, the inhibition of colon cancer cells is manifested in the inhibition of colon cancer cell viability. Further, in some preferred embodiments of this application, the inhibitory effect of SP-4 on HCT-116 colon cancer cells is determined using cell viability and cell cycle assays.

[0090] The term "cell viability" as used in this application refers to the ability of cells to maintain normal metabolism, proliferation and physiological functions under certain experimental conditions, and is usually expressed as the percentage of live cells in the total number of cells.

[0091] Optionally, when determining the cell viability of HCT-116 colorectal cancer cells, dimethyl sulfoxide (DMSO) is used as a positive control, the cell culture temperature is 37°C, the cell culture conditions are 5% CO2, and the cell culture time is 48 h; when determining the cell cycle of HCT-116 colorectal cancer cells, the cell culture temperature is 37°C, the incubation time in the dark is 30 min, and the cell culture time is 48 h.

[0092] Optionally, when measuring cell viability, the positive control group is dimethyl sulfoxide (DMSO) at a cell density of 5 × 10⁻⁶. 3 -1×10 4 Cells / well, SP-4 sample solution concentrations of 0, 200, 500, 1000, and 2000 μg / mL, CCK-8 reagent addition of 10 µL, treatment conditions of incubation at 37 ℃ in a 5% CO2 incubator for 1–4 h, and measurement wavelength of 450 nm.

[0093] Optionally, the cell cycle assay method is as follows: after treating cells with the optimal concentration of 2000 μg / mL SP-4, fix with 70% ethanol, incubate with PI staining solution in the dark for 30 min, and detect by flow cytometry (excitation wavelength 488 nm, emission wavelength 617 nm). The apoptosis assay method is as follows: Annexin V-FITC / PE and PI double staining, and flow cytometry detection of FITC / PE and PI channel fluorescence signals.

[0094] The term "cell cycle" as used in this application refers to the entire process a cell undergoes from the completion of one division to the end of the next, typically divided into four phases: G1 phase (Gap 1): a period of active cell growth and metabolism, preparing for DNA replication; S phase (Synthesis): the period of DNA synthesis, where genetic material is replicated; G2 phase (Gap 2): the period when the cell makes final preparations for division; and M phase (Mitosis): the period of mitosis, where the cell divides into two daughter cells. Propidium iodide (PI) binds to DNA, and the fluorescence intensity reflects the DNA content, distinguishing each phase of the cell cycle.

[0095] Part VII. This invention provides a polypeptide whose amino acid sequence is shown in Sequence 1 (YSDITRPG) of the sequence listing. Specifically, the polypeptide whose amino acid sequence is shown in Sequence 1 (YSDITRPG) of the sequence listing is a polypeptide with the amino acid sequence tyrosine-serine-aspartic acid-isoleucine-threonine-arginine-proline-glycine (Tyr-Ser-Asp-Ile-Thr-Arg-Pro-Gly).

[0096] The applicant further identified the structure of the target substance SP-4, laying the foundation for elucidating its structure-activity relationship. Specifically, the amino acid sequence of the target component was determined by gel permeation chromatography, amino acid analyzer, matrix-assisted laser desorption / ionization mass spectrometry, and liquid chromatography-tandem mass spectrometry.

[0097] Furthermore, in some preferred embodiments of this application, gel filtration chromatography is used to determine the molecular weight of SP-4.

[0098] The term "gel permeation chromatography" used in this application, also known as size exclusion chromatography, is an analytical technique that separates molecules based on differences in molecular size. Its principle is to utilize porous gel packing material, allowing molecules of different sizes to have different retention times as they flow through the chromatographic column.

[0099] Optionally, the mobile phase is 20 mM Tris and 100 mM NaCl at pH 8.0, the flow rate is 0.5 mL / min, the column temperature is 35 °C, and the running time is 70 min.

[0100] Furthermore, in some preferred embodiments of this application, an amino acid composition analyzer is used to determine the amino acid composition of SP-4.

[0101] The term "amino acid analyzer" as used in this application refers to an instrument specifically designed for the quantitative determination of the composition and content of amino acids in a sample. Its working principle is based on ion-exchange chromatography, which separates amino acids through differences in their interactions with the resin, and then reacts them with a colorimetric reagent (such as ninhydrin) to generate a colored compound for detection.

[0102] Optionally, the amino acid composition analyzer is configured with the following hydrolysis conditions: 6M HCl treatment at 110°C for 24 hours; a Na-type cation exchange resin column; a reaction flow rate of 10 mL / h; a buffer flow rate of 20 mL / h; a column temperature programmed at 55-65-77°C; and detection wavelengths of 570 nm and 440 nm.

[0103] Furthermore, in some preferred embodiments of this application, the SP-4 amino acid sequence is determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.

[0104] The term "matrix-assisted laser desorption / ionization time-of-flight mass spectrometer" as used in this application refers to a modular liquid chromatography purification system consisting of a pump, detector, autosampler, fraction collector, and control software, specifically designed for the separation and purification of biological macromolecules (such as proteins and peptides).

[0105] Optionally, the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis conditions are: laser wavelength of 337 nm, accelerating voltage of 20 kV, and detection mode of reflection mode.

[0106] Part 8, the present invention provides the use of the polypeptide in any of the following (1c)-(c9): (c1) In vitro antioxidant activity for non-therapeutic purposes; (c2) Preparation of antioxidant products; (c3) In vitro inhibition of Staphylococcus aureus and / or Escherichia coli for non-therapeutic purposes; (c4) Prepare disinfectants or drugs that inhibit Staphylococcus aureus and / or Escherichia coli; (c5) Preparation of angiotensin-transferase inhibitors; (c6) Prepare drugs for the prevention and / or treatment of hypertension; (c8) In vitro inhibition of colon cancer cells for non-therapeutic purposes; (c9) Prepare drugs for the prevention and / or treatment of colon cancer.

[0107] Based on the aforementioned optimal active component, the applicant further conducted tests on its antioxidant activity, antibacterial activity, angiotensin-converting enzyme inhibition rate, activity, proliferation, and apoptosis of HCT-116 colon cancer cells, and found that it has significant physiological activity.

[0108] Part 9, the present invention provides a product of any one of (d1)-(d5) below, wherein the active ingredient is the polypeptide (the polypeptide shown in sequence 1 (YSDITRPG) in the sequence listing). (d1) Antioxidant drugs and antioxidant cosmetics; (d2) Disinfectants or drugs that inhibit Staphylococcus aureus and / or Escherichia coli; (d3) Angiotensin-transferase inhibitors; (d5) Medications for treating colon cancer.

[0109] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0110] Unless otherwise specified, the methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0111] The Spirulina platensis powder used in the following examples was purchased from Dongfang Xindaze Natural Biotechnology Co., Ltd.

[0112] Example 1 Deep eutectic solvent extraction of crude spirulina polypeptide extract Step 1): Mix betaine, glycerol and water in a molar ratio of 1:2:1, and shake in a constant temperature metal bath at 85°C until clear and transparent. Cool and let stand to room temperature to obtain a deep eutectic solvent.

[0113] Step 2): Mix Spirulina platensis powder with ultrapure water at a ratio of 1:50 to prepare a homogeneous spirulina powder solution, and let it stand at 4 ℃ for 12 h. Then, place the solution in an ice-water bath and sonicate it using an ultrasonic cell disruptor. The sonication conditions were set as follows: power 700 W, 10 s intervals after 10 s of sonication, and a total treatment time of 30 min. The solution was then frozen in liquid nitrogen and thawed at room temperature, with four freeze-thaw cycles. The solution was then centrifuged at 8000 r / min at 4 ℃ for 20 min, and the supernatant was collected.

[0114] The supernatant was collected after treatment, and an appropriate amount of ammonium sulfate was added. Salting was performed stepwise to achieve a final saturation of 50% (30% → 50%), followed by standing at 4 °C for 12 h. After standing, the solution was centrifuged at 8000 r / min for 20 min at 4 °C, and the precipitate was collected and dissolved in 0.05 M PBS buffer (pH 6.86). The dissolved solution was placed in a dialysis bag and dialyzed at 4 °C to remove salts. The endpoint of dialysis was determined by titration with 0.2% barium chloride solution. After dialysis, the solution was freeze-dried under vacuum to obtain phycocyanin.

[0115] Step 3): Add DES and phycocyanin to a centrifuge tube at a ratio of 80:1 (v / m, g / mL), vortex until homogeneous, and incubate in a metal bath at 80 °C and 1000 rpm for 18 h. Then centrifuge at 4 °C and 8000 rpm for 20 min, collect the supernatant and place it in a dialysis bag with a molecular weight cutoff of 500 Da. Place the dialysis bag in ultrapure water and dialyze at 4 °C to retain spirulina peptides with a molecular weight cutoff >500 Da. Freeze-dry to obtain the spirulina peptide sample (spirulina peptide crude extract).

[0116] The extraction yield of spirulina polypeptide was calculated as: mass of crude spirulina polypeptide extract / mass of phycocyanin protein. After three replicates, the extraction yield was 68.55 ± 0.38 mg / g.

[0117] Example 2 Spirulina polypeptides were obtained by referring to the method in Example 1, except that in step 1), betaine and 1,3-propanediol were mixed in a molar ratio of 1:3 and shaken in a constant temperature metal bath at 80°C until clear and transparent. The mixture was then cooled and allowed to stand at room temperature to obtain a deep eutectic solvent.

[0118] The extraction yield was calculated to be 70.71 ± 0.7 mg / g after three replicates.

[0119] Example 3 Spirulina polypeptides were obtained by referring to the method in Example 1, except that in step 1), betaine and ethylene glycol were mixed in a molar ratio of 1:3, and shaken in a constant temperature metal bath at 80°C until clear and transparent. The mixture was then cooled and allowed to stand at room temperature to obtain a deep eutectic solvent.

[0120] The extraction yield was calculated to be 66.58 ± 0.45 mg / g after three replicates.

[0121] like Figure 1 As shown in the above embodiments, the deep eutectic solvent obtained by mixing betaine and 1,3-propanediol at a molar ratio of 1:3 yields the best extraction yield of spirulina polypeptides.

[0122] Example 4 Antioxidant activity assay and antibacterial experiment Spirulina polypeptide crude extracts extracted with three deep eutectic solvents (DES): betaine-glycerol-water (Example 1), betaine-1,3-propanediol (Example 2), and betaine-ethylene glycol (Example 3) were screened for the optimal DES extraction system by antioxidant activity determination and antibacterial experiment.

[0123] (1) Antioxidant activity test Weigh 4 mg of DPPH, dilute to 50 mL with ethanol, and prepare a solution with a concentration of 2 × 10⁻⁶ mg / mL. -4 The extract was prepared by adding 100 µL of DPPH solution and then diluting it with ethanol to a suitable concentration. The spirulina polypeptide sample was then prepared into a working solution with a concentration of 5 mg / mL. 100 µL of DPPH solution and 100 µL of working solution were added sequentially to each well of a 96-well plate. After reacting at room temperature in the dark for 30 min, the absorbance was measured at 517 nm using a microplate reader. Ax Replace 100 µL of the sample working solution with deionized water and measure its absorbance. A0 The absorbance was measured by replacing DPPH with 100 µL of ethanol. A1 Each concentration was measured in triplicate, and the clearance rate was calculated using formula (1):

[0124] Three replicates were performed. The calculated DPPH scavenging rates were 62.97±0.45% for spirulina peptides extracted with betaine-glycerol-water, 64.96±1.21% for spirulina peptides extracted with betaine-1,3-propanediol, and 60.24±1.13% for spirulina peptides extracted with betaine-ethylene glycol.

[0125] In the above embodiments, the spirulina polypeptide extracted with betaine and 1,3-propanediol exhibited the best antioxidant activity.

[0126] (2) Antibacterial test Prepare 150 mL of BHI liquid medium, LB liquid medium, and 0.85% physiological saline. After autoclaving, cool to 37 °C in a clean bench. Inoculate ceramic beads adsorbed with Staphylococcus aureus into BHI liquid medium and ceramic beads adsorbed with Escherichia coli into LB liquid medium. Incubate at 37 °C for 12 h. After incubation, take appropriate amounts of Staphylococcus aureus and Escherichia coli culture media into centrifuge tubes, centrifuge at 8000 rpm for 5 min, discard the supernatant, and collect the bacterial pellet. Resuspend the bacterial pellet in 0.85% physiological saline and mix thoroughly using a vortex mixer. Measure the OD value of the bacterial suspension at 600 nm and adjust the suspension density to 1 × 10⁻⁶. 6 -10 7 CFU / mL, prepare the bacterial suspension and refrigerate at 4°C for later use. Prepare BHI and LB solid media, autoclave them, pour them into sterile petri dishes, and then plate them for later use. At the same time, wrap 150 mL of ultrapure water and an appropriate amount of cotton swabs in aluminum foil and autoclave them together. After sterilization, air dry the cotton swabs under aseptic conditions for later use.

[0127] Spirulina peptides extracted by the three DES methods in Examples 1-3 were prepared into sample solutions with a concentration of 150 mg / mL using sterile ultrapure water. Staphylococcus aureus suspension was evenly spread onto BHI plates using sterile cotton swabs, and Escherichia coli suspension was evenly spread onto LB plates. After standing for 15 min, wells (approximately 6 mm in diameter) were punched at equal intervals on the plates, and peptide sample solutions extracted by different DES methods were added to the wells. Physiological saline was added to the center of each plate as a negative control. Three replicates were prepared for each treatment. The culture dishes were inverted and incubated in a 37 ℃ incubator for 12 h. The diameter of the inhibition zone was observed and measured, and the antibacterial effect was statistically analyzed.

[0128] like Figure 2 As shown, the inhibition zone diameter of betaine and 1,3-propanediol (Bet-PDO) against Staphylococcus aureus was approximately 0.8 cm, and that against Escherichia coli was approximately 1.8 cm. Betaine and glycerol (Bet-Gly) showed no inhibitory effect on Staphylococcus aureus, but the inhibition zone diameter against Escherichia coli was approximately 0.8 cm. Betaine and ethylene glycol (Bet-EG) showed an inhibition zone diameter of approximately 0.5 cm against Staphylococcus aureus, but no inhibitory effect against Escherichia coli. The negative control showed no inhibitory effect. The spirulina polypeptide extracted with betaine and 1,3-propanediol (Bet-PDO) exhibited the largest inhibition zone diameter against both Staphylococcus aureus and Escherichia coli, indicating that the spirulina polypeptide extracted with betaine and 1,3-propanediol showed the best antibacterial activity.

[0129] Based on the above results, betaine and 1,3-propanediol were selected as the eutectic solvents for extracting spirulina polypeptides for subsequent separation and purification experiments.

[0130] Example 5 Isolation and screening of spirulina polypeptides Step 1): Weigh 12.114 g of Tris and dissolve it in approximately 1.6 L of ultrapure water, stirring thoroughly until completely dissolved. Then, add 11.688 g of NaCl and continue stirring until completely dissolved. Add HCl solution dropwise to adjust the pH to 8.0, and bring the volume to 2 L with deionized water. Perform ultrafiltration on the buffer solution using a 0.22 μm filter membrane, followed by sonication for 30 min.

[0131] Step 2): The prepared pH 8.0 Tris-NaCl buffer (20 mM Tris, 100 mM NaCl) was passed through a Superdex™ 75 pg chromatography column at a column head pressure of 0.50 MPa and a constant flow rate of 2 mL / min, and continuously washed for 4 h to ensure the column was fully equilibrated and to remove any residual impurities. Subsequently, the Spirulina peptide sample obtained in Example 2 was dissolved in ultrapure water to prepare a peptide solution with a concentration of 200 mg / mL. This solution was filtered through a 0.22 μm filter membrane and loaded onto the equilibrated chromatography column at a constant flow rate of 2 mL / min and a column pressure of 0.50 MPa. During elution, the signal change of the elution peak was monitored in real time at a wavelength of 280 nm using a UV detector, and the eluent corresponding to the target peak was collected. 36 tubes were used, each containing 10 mL of eluent.

[0132] like Figure 3 As shown, fractions from tubes 20 to 24 were collected. SP-1 to SP-5 were collected from tube 20 (260-270 mL), tube 21 (270-280 mL), tube 22 (280-290 mL), tube 23 (290-300 mL), and tube 24 (300-310 mL), respectively. After freeze-drying, they were named the five fractions SP-1 to SP-5.

[0133] Step 4): The different components (SP-1 to SP-5) after freeze-drying were subjected to antioxidant experiments. The antioxidant activity detection method was the same as the steps in the antioxidant activity determination in Example 4. Three replicates were performed, and the calculated DPPH scavenging rates were 68.25±0.25% for SP-1, 69.23±0.45% for SP-2, 69.90±0.26% for SP-3, 70.82±0.58% for SP-4, and 70.29±0.64% for SP-5. Therefore, SP-4 exhibited the best antioxidant activity.

[0134] The freeze-dried SP-4 was further subjected to antioxidant experiments. The antioxidant activity detection method was the same as described above, except that SP-4 was prepared into sample solutions with concentrations of 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, and 8 mg / mL, respectively. Each sample was tested in triplicate, and the IC50 of SP-4 was calculated. 50 value.

[0135] like Figure 4 As shown, SP-4 was formulated into different concentrations, and the DPPH scavenging rate at different concentrations was nonlinearly fitted to obtain the IC50 of SP-4. 50 The value was 3.89 ± 0.45 mg / mL.

[0136] Step 4): The antibacterial activity detection method is carried out according to the steps in the antibacterial experiment in Example 4, except that the filter paper method is used instead of the perforation method.

[0137] like Figure 5 As shown, three replicates were performed. Calculations showed that the inhibition zone diameters of SP-1, SP-2, SP-3, SP-4, and SP-5 against Staphylococcus aureus were 0.4 cm, 0.4 cm, 0.4 cm, 0.5 cm, and 0.4 cm, respectively. The inhibition zone diameters of SP-1, SP-2, SP-3, SP-4, and SP-5 against Escherichia coli were no inhibition, no inhibition, no inhibition, 0.6 cm, and no inhibition, respectively. It can be seen that SP-4 exhibited the best antibacterial effect in the antibacterial experiments against Staphylococcus aureus and Escherichia coli, with significantly higher activity than other components and the negative control.

[0138] Similarly, SP-4 was prepared into sample solutions with concentrations of 200 mg / mL, 180 mg / mL, 170 mg / mL, 150 mg / mL, 125 mg / mL, 100 mg / mL, 70 mg / mL, and 50 mg / mL. Statistical analysis of the inhibition zone diameters against Staphylococcus aureus and Escherichia coli at different concentrations showed that the diameter of the inhibition zone of SP-4 against Staphylococcus aureus and Escherichia coli increased significantly with increasing concentration. Figure 6At a concentration of 200 mg / mL, the diameter of the inhibition zone against Staphylococcus aureus reached its maximum value (1.3±0.06 cm), and the diameter of the inhibition zone against Escherichia coli reached its maximum value (2±0.1 cm), which were significantly higher than those of the low concentration group.

[0139] As can be seen from the above, SP-4 exhibits the best antibacterial activity. Furthermore, considering its high yield (SP-4 represents the peak position in the AKTA spectrum), SP-4 was ultimately chosen as the target for subsequent activity testing and structural identification.

[0140] Example 6 Angiotensin-converting enzyme inhibition experiment Step 1): Prepare sample solutions of SP-4 obtained in Example 4 at concentrations of 1 mg / mL to 9 mg / mL using borate-borax buffer (BB Buffer). Simultaneously prepare a 6.5 mM hippuryl-histidyl-leucine (HHL) standard solution and a 0.1 U / mL angiotensin-converting enzyme solution, and prepare a 1 M HCl solution using ultrapure water.

[0141] Step 2): Prepare the angiotensin-converting enzyme reaction system, including a blank group, a sample group, and a control group. After the reaction is complete, add 150 μL of acetonitrile to each centrifuge tube to terminate the reaction, and filter the mixture through a 0.22 μm filter membrane.

[0142] Step 3): High-performance liquid chromatography (HPLC) was used to detect and quantify the generated hippuric acid. The HPLC analysis conditions were as follows: Hypersil ODS2 column (200 mm × 4.6 mm, 5 μm), mobile phase: 25% acetonitrile and 75% ultrapure water (containing 0.1% trifluoroacetic acid, TFA), flow rate: 1 mL / min, detection wavelength: 228 nm, column temperature: 30℃, injection volume: 20 μL. The formula for calculating the angiotensin-converting enzyme inhibition rate is:

[0143] In the formula, A Peak area of ​​angiotensin-converting enzyme from 0 to 2.98 min; A i - Peak areas of samples with different concentrations at 2.98 min; A 空白 Peak area of ​​blank sample at -2.98 min.

[0144] like Figure 7 As shown, SP-4 was prepared at different concentrations, and linear fitting was performed on the angiotensin-converting enzyme inhibition rate at different concentrations to obtain the IC50 of SP-4. 50The value was 6.15 mg / mL.

[0145] Example 7 Colon cancer cell inhibition test Step 1): HCT-116 colon cancer cells were injected at a dose of 5 × 10⁻⁶. 3 ~1×10 4 Cells were seeded at a density of 100 µL / well in 96-well plates, with 100 µL of culture medium added to each well. A negative control group was set up: the culture medium contained no sample solution, only the same volume of solvent (e.g., PBS); a positive control group was set up: dimethyl sulfoxide (DMSO) was added. Cells were cultured at 37 °C and 5% CO2 for 24 h to allow cell adhesion.

[0146] Replace the culture medium with different concentrations of SP-4 obtained in Example 4 (0, 200, 500, 1000, 2000 μg / mL), with at least three replicate wells for each group. Add the same volume of solvent or positive control drug to each well. Continue culturing at 37 °C and 5% CO2 for 24 h and 48 h. Add 10 µL of CCK-8 reagent to each well (avoid touching the well walls to prevent contamination). Gently shake the 96-well plate to ensure uniform distribution of CCK-8. Incubate at 37 °C and 5% CO2 for 1–4 h. Measure the absorbance (OD value) of each well at 450 nm using a microplate reader. Calculate cell viability (relative inhibition rate), using the OD value of the negative control group as 100% cell viability to analyze the effect of different concentrations of SP-4 on cell viability.

[0147] like Figure 8 As shown, the inhibitory effect of SP-4 on HCT-116 cells exhibited significant time- and concentration-dependent effects. Under 24-h treatment conditions, the lowest HCT-116 cell viability (88.07 ± 0.05%) was observed at a SP-4 concentration of 2000 μg / mL. However, under 48-h treatment conditions, the lowest HCT-116 cell viability was observed at a SP-4 concentration of 2000 μg / mL, at which point cell viability was 51.02 ± 0.03%. These results indicate that prolonged treatment time significantly enhances the cytotoxic effect of the target substance. Step 2): Seed HCT-116 cells in 96-well plates or culture flasks, treating the cells with the optimal concentrations of the four samples used for cell viability assays. The treatment time was set according to the optimal time for cell viability assays (24 h or 48 h). After treatment, aspirate the culture medium and gently wash 1-2 times with PBS. Add an appropriate amount of trypsin (without EDTA) to digest the cells. After the cells detach, add culture medium to stop the digestion. Collect the cell suspension in a centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add 1 mL of ice-cold 70% ethanol to resuspend the cells. Add ethanol slowly to avoid cell aggregation and maintain the ethanol temperature at 4 °C. Place the fixed cells in a 4 °C refrigerator for at least 2 h or overnight. After fixation, centrifuge to remove ethanol, and wash the cells twice with PBS to remove residual ethanol. Add an appropriate amount of PI staining solution (usually a PBS solution containing 50 µg / mL PI and 100 µg / mL RNase A) to resuspend the cells and incubate in the dark for 30 min (37 °C). Stained cells were analyzed using flow cytometry (excitation wavelength: 488 nm; emission wavelength: 617 nm). A DNA content histogram was plotted using flow cytometry software, selecting the DNA content (PI signal) channel. The histogram was then fitted to the cell cycle distribution to calculate the percentage of cells at each cycle stage (G1, S, G2).

[0148] like Figure 9 As shown, the proportion of cells in G1 phase was the highest (54.95±0.02%), the proportion in S phase was the lowest (28.48±0.01%), and the proportion in G2 phase was in the middle (16.56±0.01%). With the SP-4 concentration increasing from 500 µg / mL to 2000 µg / mL, the proportion of cells in G1 phase generally decreased (45.71±0.04%→43.43±0.01%), while the proportion of cells in S phase significantly increased (39.64±0.08%→44.26±0.01%), with the highest concentration group showing a 15.78% increase in S phase proportion compared to the control group. Notably, the proportion of cells in G2 phase remained relatively stable across treatment groups (12.29±0.004%-14.65±0.05%), showing a slight decrease only in the high concentration group. These results suggest that SP-4 may function by interfering with a key regulatory mechanism of the G1 / S phase transition.

[0149] Example 8 SP-4 molecular weight test Step 1): Prepare a mixed buffer solution of 20 mM Tris and 100 mM NaCl with a pH of 8.0. After preparation, use a 0.22 μm filter membrane for ultrafiltration and sonication for 30 min.

[0150] Step 2): Set the mobile phase flow rate to 0.2 mL / min and the column temperature to 35 °C, and equilibrate the column overnight. Before use, flush the column at a flow rate of 0.5 mL / min until the detector signal stabilizes.

[0151] Step 3): Dissolve SP-4 obtained in Example 4 in the mobile phase to prepare a 1 mg / mL sample solution. Centrifuge at 8000 rpm for 15 min and collect the supernatant. After filtering the supernatant through a 0.22 μm filter membrane, inject 0.2 mL into the GPC injection loop. Set the mobile phase flow rate to 0.5 mL / min, the column temperature to 35 ℃, and the run time to 70 min. Use a differential refractive index detector (RI detector) and a multi-angle laser light scattering detector (LS detector) to monitor the elution peak at a wavelength of 658 nm.

[0152] like Figure 10 As shown, SP-4 exhibited a distinct main peak between elution times of 20.0 and 40.0 min, indicating a high proportion of this component in the sample. The integration results showed that the number-average molecular weight (Mn) of the main peak was 4.267 × 10⁻⁶. 2 Da, with a weight-average molecular weight (Mw) of 8.126 × 10⁻⁶. 2 The value of Da indicates that the molecular weight of the main components of SP-4 is between 426.7 and 812.6 Da.

[0153] Example 9 SP-4 Amino Acid Types and Content Test Step 1): Accurately weigh 100 mg of the SP-4 sample obtained in Example 4 and add it to a hydrolysis tube. Add 10 mL of 6M HCl, purge the tube with nitrogen gas for 30 s and seal it. Place the tube in an oil bath at 110 °C for 24 h for hydrolysis.

[0154] Step 2): After hydrolysis, wait for the sample to cool to room temperature, filter it through a 0.45 μm filter membrane into a 50 mL volumetric flask and make up to volume.

[0155] Step 3): Take 2 mL of the diluted sample and place it on a rotary evaporator at 45 °C to remove acidity until dry. Add 2 mL of sodium citrate buffer to dissolve completely, filter through a 0.45 μm filter, and then analyze.

[0156] Step 4): The buffer flow rate was set to 20 mL / h, the reaction flow rate to 10 mL / h, the separation column to be a Na-type cation exchange resin column (200 mm × 4.6 mm, 8 μm particles), the UV detection wavelengths to be 570 nm and 440 nm, the column temperature to be programmed at 55-65-77 ℃, the reaction chamber temperature to be 138 ℃, and the injection volume to be 50 μL. The detection method was as follows: after separation in the separation column, amino acids reacted with ninhydrin. The products were detected at 570 nm using a spectrophotometer. By comparing the retention time and peak area of ​​standard amino acids, the types and contents of each amino acid in SP-4 were determined.

[0157] like Figure 11 As shown, SP-4 contains a rich variety of amino acids, and the content distribution of each amino acid varies significantly. The relative contents of Tyr (1.812 mg / g), Phe (1.73 mg / g), Arg (1.28 mg / g), Gly (1.23 mg / g), and Ala (1.06 mg / g) are higher than other amino acids in the sample, indicating that these amino acids may be the main components of SP-4. Conversely, the contents of Met (0.036 mg / g) and Pro (0.082 mg / g) are relatively low, indicating that these amino acids constitute a smaller proportion in the sample.

[0158] Example 10 Amino acid sequence testing of SP-4 Step 1): Dissolve the SP-4 peptide sample obtained in Example 4 in a mixed solvent of 50% ACN and 0.1% TFA to prepare a solution of 1 mg / mL.

[0159] Step 2): Mix the peptide solution and CHCA matrix at a 1:1 ratio, spot the mixture onto a stainless steel target plate, and allow it to crystallize naturally. Place the target plate in a MALDI-TOF-MS instrument, set the laser wavelength to 337 nm, the accelerating voltage to 20 kV, and use the reflection mode for detection.

[0160] like Figure 12 As shown, SP-4 may have a relatively complex molecular structure. The primary spectrum shows a significant molecular ion peak at m / z 907.677, indicating that the molecular weight of SP-4 is approximately 908 Da. In the secondary spectrum, the signal peak appearing in the high region of m / z = 907.481 further supports the possibility of its existence as a polymer or adduct, and the amino acid sequence of SP-4 is deduced to be YSDITRPG.

[0161] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for extracting spirulina polypeptides, characterized in that, Includes the following steps: Spirulina polypeptide source material is mixed with a deep eutectic solvent for extraction to obtain crude spirulina polypeptide extract; wherein, the deep eutectic solvent is betaine and 1,3-propanediol in a molar ratio of 1:

3. The spirulina polypeptide source material includes phycocyanin extracted from Spirulina platensis powder; The solid-liquid ratio of the spirulina polypeptide source material and the deep eutectic solvent is 1:(40-120)g / mL; The extraction was carried out under shaking conditions, at a temperature of 65–85°C, a speed of 800–1200 rpm, and a time of 6–30 h. The method further includes the following step: separating and collecting components with a molecular weight >500 Da from the spirulina polypeptide extract obtained in the extraction step.

2. The crude spirulina polypeptide extract obtained by the method of claim 1.

3. The use of the crude spirulina polypeptide extract according to claim 2 in any one of the following (a1)-(a4): (a1) In vitro antioxidant activity for non-therapeutic purposes; (a2) Preparation of antioxidant products; (a3) In vitro inhibition of Staphylococcus aureus and / or Escherichia coli for non-therapeutic purposes; (a4) Prepare disinfectants or drugs that inhibit Staphylococcus aureus and / or Escherichia coli.

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