Spirulina peptide, preparation method, product and application

Abstract

The invention belongs to the technical field of preparation of peptides, and particularly relates to a spirulina peptide, a preparation method, a product and application. The invention provides a spirulina peptide. The spirulina peptide comprises a first algae peptide, a second algae peptide, a third algae peptide, a fourth algae peptide and a fifth algae peptide. According to the preparation method of the spirulina peptide provided by the technical scheme, the neutral protease can preliminarily decompose protein in spirulina, the binding structure of the protein and polysaccharide is destroyed, and macromolecular protein is decomposed into medium-sized peptide fragments. Alkaline protease can deeply hydrolyze protein to generate the antiviral pneumonia active peptide with biological activity. And the papain can accurately regulate and control the molecular weight of the peptide fragment, so that the generated peptide fragment has relatively high water solubility and emulsifying activity, and the antiviral effect is high. Results of embodiments show that the spirulina peptide provided by the invention has the efficacy of resisting viral pneumonia.

Description

Technical Field

[0001] This invention belongs to the field of peptide preparation technology, specifically relating to a spirulina peptide, its preparation method, product, and application. Background Technology

[0002] Algae that grow in both seawater and freshwater are rich in nutrients, especially protein. They also contain various functional food components such as essential amino acids, polysaccharides, polyunsaturated fatty acids, vitamins, carotenoids, and minerals. Spirulina peptides, as substances with multiple physiological activities, hold promise for the development of products with specific functions.

[0003] Current research on the physiological functions of spirulina peptides has verified that they possess antioxidant, anti-inflammatory, lipid and glucose metabolism regulating, blood pressure lowering, and anti-cancer activities. Furthermore, studies have found that spirulina peptides can promote energy consumption in adipocytes and inhibit hepatic gluconeogenesis by activating the AMPK (adenosine monophosphate-activated protein kinase) pathway, while simultaneously increasing insulin sensitivity. This results in reduced body weight, body fat percentage, and serum glycerol levels in obese model mice, and improved insulin resistance, suggesting that small-molecule spirulina peptides can alleviate abnormalities in glucose and lipid metabolism. A research report in the international journal *Hypertension* shows that a peptide, SP6, isolated from spirulina, can dilate arteries and produce an antihypertensive effect. No reports of spirulina peptides with antiviral pneumonia effects have been found in the current technology. Summary of the Invention

[0004] The purpose of this invention is to provide a spirulina peptide that can fight viral pneumonia.

[0005] To address the aforementioned technical problems, the present invention proposes the following technical solution:

[0006] This invention provides a spirulina peptide, comprising a first spirulina peptide, a second spirulina peptide, a third spirulina peptide, a fourth spirulina peptide, and a fifth spirulina peptide; the first spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 10; the second spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 4; the third spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 11; the fourth spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 8; and the fifth spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 3.

[0007] Preferably, the first algal peptide is a first polypeptide and / or a tenth polypeptide; the amino acid sequence of the first polypeptide is as shown in SEQ ID No. 1, and the amino acid sequence of the tenth polypeptide is as shown in SEQ ID No. 10;

[0008] The second algal peptide is a second polypeptide and / or a fourth polypeptide, the amino acid sequence of the second polypeptide is shown in SEQ ID No. 2, and the amino acid sequence of the fourth polypeptide is shown in SEQ ID No. 4;

[0009] The third phycopeptide is one or more of the fifth polypeptide, the seventh polypeptide, the ninth polypeptide, and the eleventh polypeptide; the amino acid sequence of the fifth polypeptide is shown in SEQ ID No. 5, the amino acid sequence of the seventh polypeptide is shown in SEQ ID No. 7, the amino acid sequence of the ninth polypeptide is shown in SEQ ID No. 9, and the amino acid sequence of the eleventh polypeptide is shown in SEQ ID No. 11.

[0010] The fourth phycopeptide is the sixth polypeptide and / or the eighth polypeptide; the amino acid sequence of the sixth polypeptide is shown in SEQ ID No. 6, and the amino acid sequence of the eighth polypeptide is shown in SEQ ID No. 8;

[0011] The fifth algal peptide is the third polypeptide, and the amino acid sequence of the third polypeptide is shown in SEQ ID No. 3.

[0012] This invention provides a method for preparing the spirulina peptide described in the above technical solution, comprising the following steps:

[0013] Spirulina was subjected to a first and a second enzymatic hydrolysis to obtain spirulina peptides. The enzymes used in the first enzymatic hydrolysis included neutral protease, and the enzymes used in the second enzymatic hydrolysis included alkaline protease and papain.

[0014] Preferably, the temperature of the first enzymatic hydrolysis is 35-40°C, and the time of the first enzymatic hydrolysis is 55-65 min; the amount of neutral protease added is 0.018%-0.022% of the mass of Spirulina.

[0015] Preferably, after the first enzymatic hydrolysis, homogenization is further performed at a pressure of 33-38 kPa, and the homogenization is performed once.

[0016] Preferably, the enzyme used in the second enzymatic hydrolysis is added in several stages, including an initial addition and a final addition; the time interval between the initial addition and the final addition is ≤30 min.

[0017] The enzymes added for the first time include alkaline protease and papain;

[0018] The enzyme added in the last addition includes alkaline protease; the enzymatic hydrolysis time is started after the last addition, and the enzymatic hydrolysis time is 4-5 hours.

[0019] Preferably, the mass of the enzyme added for the first time is 0.23% to 0.27% of the mass of spirulina; and the mass of the enzyme added for the last time is 0.07% to 0.09% of the mass of spirulina.

[0020] Preferably, the temperature of the second enzymatic hydrolysis is 50-55°C, and the pH is 8.0-8.5.

[0021] Preferably, the enzymatic hydrolysis is followed by purification and drying.

[0022] This invention provides the application of the spirulina peptide described in the above technical solution or the spirulina peptide prepared by the preparation method described in the above technical solution in the preparation of products for antiviral pneumonia.

[0023] This invention provides an antiviral pneumonia product, comprising the spirulina peptide described in the above technical solution or the spirulina peptide prepared by the preparation method described in the above technical solution.

[0024] The beneficial effects of this invention are as follows: This invention provides a spirulina peptide, comprising a first spirulina peptide, a second spirulina peptide, a third spirulina peptide, a fourth spirulina peptide, and a fifth spirulina peptide; the first spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 10; the second spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 4; the third spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 11; the fourth spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 8; and the fifth spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 3. The spirulina peptide of this invention has antiviral pneumonia efficacy.

[0025] This invention provides a method for preparing the spirulina peptides described in the above-mentioned technical solution. The neutral protease can initially decompose the proteins in spirulina, disrupting the binding structure between proteins and polysaccharides within spirulina cells. It breaks down large proteins such as phycocyanin and chlorophyll-binding proteins into medium-sized peptides with a molecular weight of approximately 1000–5000 Da, providing substrates for subsequent enzymatic hydrolysis. Simultaneously, the neutral protease can also promote cell wall disruption in spirulina, decomposing proteins in the cell wall, thereby disrupting the cell wall structure and releasing intracellular substances.

[0026] Alkaline proteases can deeply hydrolyze proteins, breaking down medium-sized peptides into smaller peptides with a molecular weight of <1000 Da, thus improving protein digestibility. Their specific cleavage of peptide bonds near phenylalanine and tyrosine makes it easier to generate bioactive peptides, promoting the production of antiviral pneumonia active peptides.

[0027] Furthermore, papain can precisely regulate the molecular weight of peptides, preferentially cleaving the carboxyl side peptide bonds of positively charged amino acids (such as arginine, lysine, and phenylalanine), improving the functional properties of the product, giving the hydrolyzed peptides higher water solubility and emulsifying activity, and enhancing the antiviral effect.

[0028] The results of the examples show that the spirulina peptide provided by the present invention has the effect of antiviral pneumonia. Attached Figure Description

[0029] Figure 1 The ultraviolet chromatogram (215 nm) and base peak ion chromatogram of the spirulina peptide prepared in Example 1;

[0030] Figure 2 Typical fluorescence images of neutrophil numbers in the swim bladder of zebrafish after different treatment groups in Example 2;

[0031] Figure 3 The bar graph shows the number of neutrophils in the swim bladder of zebrafish after different treatment groups in Example 2. Compared with the model control group, **P<0.01, ***P<0.001;

[0032] Figure 4 The graph shows the antiviral efficacy of different treatment groups in Example 2 against pneumonia. Detailed Implementation

[0033] This invention provides a spirulina peptide, comprising a first spirulina peptide, a second spirulina peptide, a third spirulina peptide, a fourth spirulina peptide, and a fifth spirulina peptide; the first spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 10; the second spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 4; the third spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 11; the fourth spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 8; and the fifth spirulina peptide comprises the amino acid sequence shown in SEQ ID No. 3.

[0034] As an optional implementation, the first algal peptide of the present invention is a first polypeptide and / or a tenth polypeptide; the amino acid sequence of the first polypeptide is as shown in SEQ ID No. 1, and the amino acid sequence of the tenth polypeptide is as shown in SEQ ID No. 10;

[0035] The second algal peptide is a second polypeptide and / or a fourth polypeptide, the amino acid sequence of the second polypeptide is shown in SEQ ID No. 2, and the amino acid sequence of the fourth polypeptide is shown in SEQ ID No. 4;

[0036] The third phycopeptide is one or more of the fifth polypeptide, the seventh polypeptide, the ninth polypeptide, and the eleventh polypeptide; the amino acid sequence of the fifth polypeptide is shown in SEQ ID No. 5, the amino acid sequence of the seventh polypeptide is shown in SEQ ID No. 7, the amino acid sequence of the ninth polypeptide is shown in SEQ ID No. 9, and the amino acid sequence of the eleventh polypeptide is shown in SEQ ID No. 11.

[0037] The fourth phycopeptide is the sixth polypeptide and / or the eighth polypeptide; the amino acid sequence of the sixth polypeptide is shown in SEQ ID No. 6, and the amino acid sequence of the eighth polypeptide is shown in SEQ ID No. 8;

[0038] The fifth algal peptide is the third polypeptide, and the amino acid sequence of the third polypeptide is shown in SEQ ID No. 3.

[0039] The spirulina peptide of this invention is a mixture of multiple peptide segments, rich in glutamic acid, aspartic acid, leucine, lysine, etc., with essential amino acids accounting for approximately 40%, close to the ideal protein pattern recommended by the Food and Agriculture Organization of the United Nations and the World Health Organization. The molecular weight distribution of the spirulina peptide is relatively broad, typically ranging from 100 to 5000 Da, with most <1000 Da. The functional peptide segments of the spirulina peptide of this invention are the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and eleventh peptides, which exert antiviral pneumonia efficacy.

[0040] Spirulina peptides are either short peptides or relatively long polypeptides. Due to enzymatic hydrolysis, the natural protein conformation is disrupted, resulting in short peptides such as dipeptides and tripeptides that typically have linear structures without complex higher-order conformations, making them easier to absorb. Longer polypeptides, on the other hand, can form simple secondary structures, lacking the original tertiary and quaternary structures of spirulina proteins. These secondary structure polypeptides are more easily absorbed and exert their biological effects.

[0041] This invention provides a method for preparing the spirulina peptide described in the above technical solution, comprising the following steps:

[0042] Spirulina was subjected to a first and a second enzymatic hydrolysis to obtain spirulina peptides. The enzymes used in the first enzymatic hydrolysis included neutral protease, and the enzymes used in the second enzymatic hydrolysis included alkaline protease and papain.

[0043] As an optional implementation, the application form of spirulina described in this invention includes spirulina powder. Compared to spirulina, spirulina powder is more suitable for industrial production, increasing the contact area during enzymatic hydrolysis and improving the hydrolysis effect. This invention does not have specific limitations on the source of the spirulina powder; conventional products are acceptable. This invention also does not have specific limitations on the preparation method and mesh size of the spirulina powder; conventional methods are acceptable.

[0044] As an optional embodiment, the present invention mixes the spirulina powder with water to form a slurry, thereby obtaining a mixture. As an optional embodiment, the mass ratio of the spirulina powder to water is 1:(7-9), more preferably 1:8. The water used in the present invention is preferably deionized water obtained through reverse osmosis treatment. As an optional embodiment, the mixing method of the present invention includes stirring. The present invention does not have specific limitations on the stirring parameters; conventional methods can be used.

[0045] As an optional embodiment, the present invention performs a first enzymatic hydrolysis and a second enzymatic hydrolysis on the mixture to obtain an enzymatic hydrolysate. The enzyme used in the first enzymatic hydrolysis of the present invention includes a neutral protease. The present invention performs the first enzymatic hydrolysis after mixing the mixture and the neutral protease. Before adding the neutral protease, it is preferable to heat the mixture to the temperature for the first enzymatic hydrolysis. The temperature for the first enzymatic hydrolysis of the present invention is 35–40°C, more preferably 36–38°C, and more preferably 37°C; the hydrolysis time is preferably 55–65 min, more preferably 58–62 min, and more preferably 60 min. The enzyme activity of the neutral protease of the present invention is preferably 1.4 million–1.6 million U / g, and more preferably 1.5 million U / g. The present invention does not have a special limitation on the source of the neutral protease; conventional products are acceptable. As an optional embodiment, the mixing method of the present invention includes stirring.

[0046] The amount of neutral protease added in this invention is 0.018% to 0.022% of the mass of spirulina, more preferably 0.02%. The timing, temperature, and neutral protease settings of the first enzymatic hydrolysis can disrupt the protein structure in spirulina, preferentially hydrolyzing the surface of protein molecules or loosely structured peptide bonds, breaking the dense structure of large protein molecules, and exposing the previously encapsulated internal peptide bonds. This provides more action sites for the subsequently added alkaline protease and papain, allowing them to more focusedly act on recalcitrant protein components, reducing subsequent enzymatic hydrolysis resistance, and improving the overall targeting and efficiency of the enzymatic hydrolysis.

[0047] The neutral protease described in this invention can initially break down proteins, disrupt the binding structure between proteins and polysaccharides in spirulina cells, and decompose large molecular proteins such as phycocyanin and chlorophyll-binding proteins into medium-sized peptides.

[0048] As an optional implementation, the present invention further includes homogenizing the product obtained after the first enzymatic hydrolysis to obtain a homogenate. The homogenization pressure is 33–38 kPa, more preferably 35 kPa, and the homogenization is performed once. Homogenization can uniformly disperse the hydrolysate, promoting the subsequent second enzymatic hydrolysis.

[0049] As an optional implementation, the present invention performs a second enzymatic hydrolysis on the homogenate.

[0050] The enzymes used in the second enzymatic hydrolysis of this invention include alkaline protease and papain. Before adding the alkaline protease and papain, it is preferable to heat the homogenate and adjust its pH. The heating to the temperature for the second enzymatic hydrolysis is 50–55°C, or 51–54°C, more preferably 53°C; the pH is adjusted to the pH for the second enzymatic hydrolysis, which is 8.0–8.5. Adjusting the temperature and pH can promote the second enzymatic hydrolysis.

[0051] In this invention, the homogenate, alkaline protease, and papain are mixed and then subjected to a second enzymatic hydrolysis. As an optional embodiment, the mixing method described in this invention includes stirring.

[0052] As an optional implementation, the enzyme used in the second enzymatic hydrolysis of this invention is added in batches, including an initial addition and a final addition; the time interval between the initial and final additions is ≤30 min. In the initial stage of enzymatic hydrolysis, the substrate concentration is usually high. If alkaline protease is added all at once, excess substrate molecules will bind to the active site of the enzyme, failing to effectively convert into product and hindering the normal reaction of the enzyme with other substrates, thus reducing catalytic efficiency. Therefore, batch addition is considered to reduce inhibition and maintain the enzyme's highly efficient catalytic state. Proteases gradually become inactive during the reaction due to changes in temperature and pH or self-degradation. Enzymes added all at once may lose activity before the substrate is completely converted, resulting in substrate residue. Batch addition allows for timely replenishment of fresh enzyme, extending the effective action time of the enzyme and ensuring that more substrate is decomposed. The catalytic reaction of alkaline protease may be accompanied by side reactions, such as excessive hydrolysis producing bitter peptides. A single high concentration of enzyme can lead to an excessively fast reaction rate, excessive local product accumulation, and increased risk of side reactions. Therefore, batch addition is adopted to steadily control the reaction rate, making product formation more uniform, reducing by-products, and thus improving the quality of the enzymatic hydrolysis product. The time interval between the first and last additions described in this invention is ≤30 minutes. This is to match the enzyme's catalytic cycle and balance the substrate consumption rate, preventing the enzyme from becoming idle due to insufficient substrate or its own inactivation, thus maximizing enzyme utilization. It also avoids rushed operations and increased errors caused by excessively short intervals.

[0053] As an optional implementation, the enzymes added initially in this invention include alkaline protease and papain; the mass of the enzymes added initially is 0.23% to 0.27% of the mass of Spirulina, more preferably 0.25%; a larger mass of enzymes added initially allows for rapid initiation of the enzymatic hydrolysis reaction, matching the high substrate concentration, avoiding the enzyme becoming a rate-limiting factor, and improving the hydrolysis efficiency. The mass ratio of alkaline protease to papain during the initial addition is (2-2.4):0.5, preferably 2.0:0.5. Setting this mass ratio of alkaline protease to papain can improve the hydrolysis efficiency of Spirulina. The enzyme activity of the alkaline protease in this invention is preferably 230-250 U / g, more preferably 2.4 million U / g. This invention does not have a special limitation on the source of the alkaline protease; conventional products are acceptable.

[0054] As an optional implementation, the papain of the present invention has an enzyme activity of 900,000 to 1,100,000 U / g, more preferably 1,000,000 U / g. The present invention does not have a particular limitation on the source of the papain; conventional products are acceptable.

[0055] The enzyme added in the final stage of this invention includes alkaline protease. Preferably, the neutral protease is added all at once during the initial addition, while the alkaline protease is added in two stages. This arrangement extends the effective action time of the enzyme, ensuring that more substrate is broken down.

[0056] The neutral protease described in this invention has an enzyme activity of 1.4 million to 1.6 million U / g, more preferably 1.5 million U / g. This invention does not have any particular limitation on the source of the neutral protease; conventional products are acceptable.

[0057] As an optional implementation, the mass of the enzyme added at the last stage of the present invention is 0.07% to 0.09% of the mass of spirulina, more preferably 0.08%. Setting the mass of the enzyme added at the last stage can avoid reaction stagnation or slowness due to insufficient enzyme quantity, and shorten the overall enzymatic hydrolysis time.

[0058] As an optional implementation, the enzymatic hydrolysis time is started after the last addition of the present invention, and the enzymatic hydrolysis time is 4 to 5 hours, more preferably 4 hours.

[0059] This invention mixes solid materials such as spirulina powder and enzyme preparations with water to form a slurry. The mixture is then stirred and homogenized to ensure uniform dispersion, increasing the substrate contact area and avoiding incomplete enzymatic hydrolysis caused by raw material clumping. This improves the degree of hydrolysis and yield.

[0060] After the second enzymatic hydrolysis, an enzymatic hydrolysate is obtained. As an optional implementation, the present invention inactivates and purifies the enzymatic hydrolysate to obtain spirulina peptides. The present invention does not have specific limitations on the parameters for enzyme inactivation; conventional methods are acceptable. In a specific embodiment of the present invention, the enzyme inactivation temperature is 80°C, and the time is 2–3 minutes. As an optional implementation, the purification of the present invention includes sequential ceramic membrane filtration, nanofiltration, concentration, and adsorption. As an optional implementation, the diameter of the ceramic membrane is 0.1 μm. Ceramic membrane filtration can remove impurities and particles, initially improving purity. The pore size of the nanofiltration membrane of the present invention is 1–2 nm. Nanofiltration of the present invention can remove salt and water from the filtrate, improving the purity of the spirulina peptides. After ceramic membrane filtration and nanofiltration, a filtrate is obtained. The present invention preferably concentrates and sterilizes the filtrate. The present invention concentrates the volume of the filtrate to 24%–30% of its original volume. The sterilization method of the present invention includes high-pressure steam sterilization. The sterilization temperature of this invention is 130–135°C, and the time is 20–30 min. This invention involves adsorption treatment of the sterilized concentrate. As an optional embodiment, the adsorption method includes activated carbon adsorption. Activated carbon adsorption can remove pigments and odors from the enzymatic hydrolysate, thereby purifying the target product and decolorizing it to improve transparency. The amount of activated carbon added in this invention is 2.5%–5% of the mass of the spirulina powder, more preferably 4%. The activated carbon adsorption temperature of this invention is 60°C; the activated carbon adsorption time is 60 min. After activated carbon adsorption, the resulting product is further subjected to plate and frame filtration.

[0061] Plate and frame filtration is used to separate activated carbon and the concentrated spirulina peptide solution, while removing other solid impurities, thereby improving the clarity and purity of the spirulina peptide solution. The purification method of this invention can improve the purity of spirulina peptides. The spirulina peptides prepared by this invention have high purity and good antiviral pneumonia effects.

[0062] As an optional implementation, the purification process of this invention further includes drying the purified product. The drying method includes spray drying. The inlet air temperature for spray drying in this invention is 180–200°C, and the outlet air temperature is 80–90°C. Spray drying continues until a powdery substance is obtained.

[0063] After drying, spirulina peptide powder is obtained. The spirulina peptide powder of this invention is white to yellowish in appearance and free of lumps. Spirulina peptide powder has good solubility, readily dissolving in water to form a clear solution; however, it is hygroscopic and requires sealed storage. Spirulina peptide powder exhibits good thermal stability and also possesses the characteristic odor of protein hydrolysates.

[0064] This invention provides the application of the spirulina peptide described in the above technical solution or the spirulina peptide prepared by the preparation method described in the above technical solution in antiviral pneumonia.

[0065] This invention utilizes experiments on zebrafish strains with green fluorescent neutrophil MPX strains to verify that spirulina peptides can reduce the number of neutrophils and have an antiviral pneumonia effect.

[0066] This invention provides an antiviral pneumonia product, comprising the spirulina peptide described in the above-described technical solution or the spirulina peptide prepared by the preparation method described in the above-described technical solution. This invention does not impose any particular limitation on the preparation method and type of the product; conventional methods can be used.

[0067] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1: Preparation of Spirulina Peptides

[0069] 1. Preparation: Mix 1000 kg of spirulina powder and 8000 kg of water, heat to 37°C, add 0.2 kg of neutral protease, hydrolyze for 1 hour, then homogenize once at a pressure of 35 kPa to obtain homogenized material.

[0070] 2. Enzymatic hydrolysis: Heat the homogenate to 50–55°C, adjust the pH to 8.0–8.5, and then add the enzyme in two batches. First, add 2.0 kg of alkaline protease and 0.5 kg of papain. Then, add 0.8 kg of alkaline protease in the second batch within 30 minutes. Start timing the hydrolysis after the second enzyme addition and perform hydrolysis for 4 hours.

[0071] The added neutral protease has an activity of 1.5 million U / g, the added alkaline protease has an activity of 2.4 million U / g, and the added papain has an activity of 1 million U / g.

[0072] 3. Enzyme inactivation: After enzymatic hydrolysis, enzyme inactivation is performed. The specific method for enzyme inactivation is as follows: steam is introduced into the enzymatic hydrolysis tank and heated to 80°C, and maintained for 2-3 minutes to deactivate papain and alkaline protease, thereby terminating the enzymatic hydrolysis reaction and obtaining the enzymatic hydrolysate.

[0073] 4. Pass the enzymatic hydrolysate through a 0.1μm ceramic membrane to remove impurities and particles, thereby initially improving the purity and obtaining the first filtrate.

[0074] 5. The first filtrate is nanofiltered using a nanofiltration membrane to remove salt and water, and then concentrated to 24%–30% of the volume of the first filtrate to obtain a concentrated solution.

[0075] 6. The concentrate is sterilized by high-pressure steam, using moist heat to penetrate the material and kill microorganisms. Sterilization temperature: 130-135℃, sterilization time: 20-30 minutes.

[0076] 7. Activated Carbon Adsorption: Activated carbon was added to the sterilized concentrate at 4% of the spirulina powder mass. The solution was then incubated at 60℃ for 60 minutes and filtered using a plate and frame filter to obtain the second filtrate. Plate and frame filtration separated the activated carbon from the concentrate, while also removing other solid impurities to improve the clarity and purity of the solution. The second filtrate, i.e., the spirulina polypeptide mixture, was analyzed by LC-MS / MS at Suzhou Kexin Kangheng Pharmaceutical Technology Co., Ltd. The results of the baseline ion chromatogram and the UV chromatogram detected at 215 nm are shown below. Figure 1 .according to Figure 1 The molecular weight, charge, and retention information of the spirulina peptide mixture were obtained. Secondary mass spectrometry analysis of the spirulina peptide mixture yielded secondary fragment ions. After database matching, a total of 472 spirulina peptides were identified. The database was the Spirulina protein database downloaded from NCBI. The spirulina peptides were mainly pentapeptides to eicoseptides, with molecular weights ranging from 500 to 2000 Da and charges from 1 to 4. Octapeptides accounted for the highest proportion, reaching 19.9% ​​with 94 peptides; followed by nonapeptides, with 89 peptides, accounting for 18.9%.

[0077] Analysis revealed that the Spirulina polypeptide mixture contained a first polypeptide, a second polypeptide, a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide, a seventh polypeptide, an eighth polypeptide, a ninth polypeptide, a tenth polypeptide, and an eleventh polypeptide. The amino acid sequence of the first polypeptide was LGKMPGDDWQKFA (SEQ ID No. 1), the amino acid sequence of the second polypeptide was FDVDWSDYQ (SEQ ID No. 2), the amino acid sequence of the third polypeptide was FLDDWEIPHL (SEQ ID No. 3), the amino acid sequence of the fourth polypeptide was DVDWSDYQ (SEQ ID No. 4), the amino acid sequence of the fifth polypeptide was QDWASMPGVMPV (SEQ ID No. 5), the amino acid sequence of the sixth polypeptide was YYGEDEDWRFT (SEQ ID No. 6), the amino acid sequence of the seventh polypeptide was QDWASMPGVMPVA (SEQ ID No. 7), the amino acid sequence of the eighth polypeptide was YYGEDEDWRF (SEQ ID No. 8), and the amino acid sequence of the ninth polypeptide was QDWASMPGVM (SEQ ID No. 1). No. 9), the amino acid sequence of the tenth polypeptide is GKMPGDDWQKF (SEQ ID No. 10), and the amino acid sequence of the eleventh polypeptide is QDWASMPGV (SEQ ID No. 11).

[0078] 8. Spray drying: The second filtrate is spray dried to obtain a solid powder, namely spirulina peptide powder. The inlet air temperature for spray drying is 180 - 200 °C, and the outlet air temperature is 80 - 90 °C.

[0079] Example 2 Verification of the efficacy against viral pneumonia

[0080] 1 Detection materials

[0081] 1.1 Sample detection configuration information

[0082] The spirulina peptide solid powder prepared in Example 1 is formulated into a 2.00 mg / ml spirulina peptide stock solution with standard dilution water and used immediately after preparation.

[0083] Positive control: Dexamethasone acetate, white powder, batch number B1828095, Shanghai Aladdin Biochemical Technology Co., Ltd., stored in a cool and dark place. It is formulated into a 100 mM stock solution with DMSO and stored at -20 °C.

[0084] 1.2 Experimental animals

[0085] Transgenic neutrophil green fluorescent zebrafish (MPX strain) are provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. All zebrafish are raised in fish-raising water at 28 °C. The water quality is: 200 mg of instant sea salt is added to every 1 L of reverse osmosis water, the conductivity is 450 - 550 μS / cm; the pH is 6.5 - 8.5; the hardness is 50 - 100 mg / L CaCO3. The license number for the use of experimental animals is: SYXK(Zhe)2022 - 0004, and the feeding management meets the requirements of international AAALAC certification (certification number: 001458).

[0086] Transgenic neutrophil green fluorescent zebrafish (MPX strain) are fertilized by natural paired mating. Zebrafish at 5 days post-fertilization (5 dpf) are used for the determination of the maximum non-toxic concentration (MTC) and efficacy evaluation of the sample against viral pneumonia without death and other toxic effects.

[0087] 1.3 Instruments, consumables and reagents

[0088] Olympus stereomicroscope (SZX7, OLYMPUS, Japan);

[0089] CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China);

[0090] Microinjector (IM300, Narishige, Japan);

[0091] Pulling needle instrument (PC-10, Narishige, Japan);

[0092] Nikon AZ100 multi-function zoom stereomicroscope imported from Japan;

[0093] Precision electronic balance (CP214, OHAUS, USA);

[0094] 6-well plate (Belamb Biotechnology (Hangzhou) Co., Ltd., China).

[0095] Methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China);

[0096] Dimethyl sulfoxide (DMSO, lot number BCCD8942, Sigma, Switzerland);

[0097] Poly(I:C) (lot number 1-KMS-178-2, trc, Canada).

[0098] 2. Detection Method

[0099] 2.1 MTC Measurement

[0100] 5-day-first-flush (dpf) transgenic neutrophilic green fluorescent zebrafish (MPX strain) were randomly selected and placed in 6-well plates. Each well (experimental group) contained 15 zebrafish, with a volume of 3 mL per well. The experimental groups were designated as treatments 1 through 6, with spirulina peptide stock solution added to achieve final concentrations of 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, and 2000 μg / mL, respectively (see Table 1). A normal control group was also included, without the addition of spirulina peptide stock solution, and otherwise identical to the experimental groups. After treatment at 28℃ for 3 hours, the median concentration (MTC) of spirulina peptide in normal zebrafish was measured. The MTC of spirulina peptide was found to be 2000 μg / mL.

[0101] Table 1. Results of the concentration exploration experiment for evaluating the antiviral efficacy of spirulina peptides against pneumonia (n=15)

[0102]

[0103]

[0104] 2.2 Evaluation of antiviral efficacy against pneumonia

[0105] 5-day-first-flush (dpf) transgenic neutrophil-positive green fluorescent zebrafish (MPX strain) were randomly selected and placed in 6-well plates, with 15 zebrafish treated in each well. Except for the normal control group, all experimental groups underwent swim bladder injection of Poly(I:C) to establish a zebrafish viral pneumonia model, followed by treatment with the drug.

[0106] Normal control group: No Poly(I:C) injection or drug administration; 3 mL per well, 5 wells in total.

[0107] Model control group: injected with Poly(I:C), no other drugs were administered; each well contained 3 mL, for a total of 5 wells.

[0108] Positive control group: Poly(I:C) was injected, followed by administration of dexamethasone acetate, with the final concentration of dexamethasone acetate being 100 μM after administration; each well had a volume of 3 mL, for a total of 5 wells.

[0109] Spirulina peptide group: Poly(I:C) was injected to administer different concentrations of spirulina peptides. The final concentrations of spirulina peptides after administration were 500 μg / mL, 1000 μg / mL, and 2000 μg / mL. The concentration data are shown in Table 2. Each well had a volume of 3 mL, for a total of 5 wells.

[0110] Soybean peptide group: Same as spirulina peptide group, the only difference is that it is replaced with soybean peptide.

[0111] Soybean peptides were prepared according to the methods described in paragraphs

[31] to

[37] of CN111707756A, a method for evaluating the stability of the production process of debittered and decolorized soybean peptides.

[0112] Whey peptide group: Same as spirulina peptide group, the only difference is that it is replaced with whey peptide.

[0113] Whey peptide enzymatic hydrolysis process:

[0114] Step 1: Add 400ml of deionized water, heat to 55℃, adjust pH to 8.5, then add 1% (0.32mL) of soybean peptide-specific alkaline protease, and slowly add 32g of milk protein isolate.

[0115] Detect the pH and adjust the pH value to 7.0-7.3. Add trypsin at 1% (0.32g) of the milk protein isolate. After half an hour, add soybean peptide-specific flavor protease at 1% (0.32ml) of the milk protein isolate. Add aminopeptidase at 1% (0.32g) of the milk protein isolate.

[0116] Enzymatic hydrolysis time: 6 hours in total, with continuous stirring during the process.

[0117] Casein peptide group: Same as spirulina peptide group, the only difference being that it is replaced with casein peptide. Casein peptide is prepared according to the method described in paragraphs

[75] to

[81] of CN112409470A A casein peptide with sleep-improving effect and its preparation method and application.

[0118] After treatment at 28℃ for 3 hours, eight zebrafish were randomly selected from each treatment group and photographed under a fluorescence microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software to analyze the number of neutrophils in the inflamed areas of the zebrafish swim bladder. The statistical analysis results of this index were used to evaluate the antiviral efficacy of spirulina peptides against pneumonia. Antiviral pneumonia efficacy (%) = (Neutrophil count in the model control group - Neutrophil count in the experimental group) / Neutrophil count in the model control group.

[0119] Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and a p-value < 0.05 was considered statistically significant. Results are shown in Table 2 and... Figures 2-4 It can be seen that spirulina peptides have antiviral pneumonia efficacy. In the soybean peptide experimental group, at a concentration of 500 μg / mL, the neutrophil count (14.9±1.14) was close to that of the model control group (17.0±0.598). At 1000 μg / mL (10.5±0.845) and 2000 μg / mL (9.38±0.680), the count decreased significantly, and the decrease became more pronounced with increasing concentration. In terms of antiviral pneumonia efficacy, the efficacy was 13% at 500 μg / mL, 38% at 1000 μg / mL, and 45% at 2000 μg / mL. The efficacy increased with increasing concentration, consistent with the decreasing trend of neutrophil count, indicating that the high concentration (2000 μg / mL) of soybean peptides had a prominent effect.

[0120] In the whey peptide experimental group, the neutrophil count was 14.9±1.11 at 500 μg / mL, close to that of the model group; it was significantly reduced at 1000 μg / mL (11.6±0.263) and 2000 μg / mL (10.4±1.76); the antiviral pneumonia efficacy was 13% at 500 μg / mL, 32% at 1000 μg / mL, and 39% at 2000 μg / mL. The efficacy increased with increasing concentration and decreasing neutrophil count, indicating that whey peptide at 2000 μg / mL had a better effect.

[0121] In the spirulina peptide experimental group, the neutrophil count was 12.3±0.796 at 500 μg / mL, and continuously decreased at 1000 μg / mL (10.3±0.620) and 2000 μg / mL (10.1±0.549), with a larger decrease at medium and high concentrations (1000 and 2000 μg / mL). The antiviral pneumonia efficacy was 28% at 500 μg / mL, and 40% at both 1000 and 2000 μg / mL. The efficacy was stable and relatively high at medium and high concentrations, indicating that the composition can exert an antiviral pneumonia effect at medium and high concentrations.

[0122] In the casein peptide experimental group, the efficacy was 11.3±0.996 at 500μg / mL, and fluctuated at 12.1±0.549 at 1000μg / mL and 12.3±1.06 at 2000μg / mL. Overall, it was lower than that of the model group, but the decrease was not as stable as that of other peptides. The efficacy was 34% at 500μg / mL, 29% at 1000μg / mL, and 28% at 2000μg / mL, showing a trend of decreasing efficacy with increasing concentration. The low concentration (500μg / mL) had a relatively certain effect.

[0123] The positive control group (dexamethasone acetate) had a concentration of 11.9 ± 1.14 at 100 μM, with an antiviral pneumonia efficacy of 30%, which can be used as a reference. Some peptides (such as soybean peptide 2000 μg / mL, spirulina peptide 1000, 2000 μg / mL, etc.) are superior to or close to it in efficacy, demonstrating the potential of peptide substances.

[0124] In summary, the experimental results of different peptides show that soybean peptides (high concentration), whey peptides (high concentration), and spirulina peptides (medium and high concentrations) have good antiviral pneumonia effects.

[0125] Table 2. Experimental results evaluating the antiviral efficacy of spirulina peptides against pneumonia (n=8)

[0126]

[0127] Note: Compared with the model control group, **P<0.01, ***P<0.001.

[0128] In summary, this invention utilizes the green fluorescent neutrophil MPX strain of zebrafish to verify that spirulina peptides can reduce the number of neutrophils and have an antiviral pneumonia effect.

[0129] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A spirulina peptide, characterized in that, The spirulina peptides include a first spirulina peptide, a second spirulina peptide, a third spirulina peptide, a fourth spirulina peptide, and a fifth spirulina peptide; the first spirulina peptide includes the amino acid sequence shown in SEQ ID No. 10; the second spirulina peptide includes the amino acid sequence shown in SEQ ID No. 4; the third spirulina peptide includes the amino acid sequence shown in SEQ ID No. 11; the fourth spirulina peptide includes the amino acid sequence shown in SEQ ID No. 8; and the fifth spirulina peptide includes the amino acid sequence shown in SEQ ID No.

3.

2. The spirulina peptide according to claim 1, characterized in that, The first algal peptide is a first polypeptide and / or a tenth polypeptide; the amino acid sequence of the first polypeptide is shown in SEQ ID No. 1, and the amino acid sequence of the tenth polypeptide is shown in SEQ ID No. 10; The second algal peptide is a second polypeptide and / or a fourth polypeptide, the amino acid sequence of the second polypeptide is shown in SEQ ID No. 2, and the amino acid sequence of the fourth polypeptide is shown in SEQ ID No. 4; The third phycopeptide is one or more of the fifth polypeptide, the seventh polypeptide, the ninth polypeptide, and the eleventh polypeptide; the amino acid sequence of the fifth polypeptide is shown in SEQ ID No. 5, the amino acid sequence of the seventh polypeptide is shown in SEQ ID No. 7, the amino acid sequence of the ninth polypeptide is shown in SEQ ID No. 9, and the amino acid sequence of the eleventh polypeptide is shown in SEQ ID No.

11. The fourth phycopeptide is the sixth polypeptide and / or the eighth polypeptide; the amino acid sequence of the sixth polypeptide is shown in SEQ ID No. 6, and the amino acid sequence of the eighth polypeptide is shown in SEQ ID No. 8; The fifth algal peptide is the third polypeptide, and the amino acid sequence of the third polypeptide is shown in SEQ ID No.

3.

3. The method for preparing the spirulina peptide according to claim 1 or 2, characterized in that, Includes the following steps: Spirulina was subjected to a first and a second enzymatic hydrolysis to obtain spirulina peptides. The enzymes used in the first enzymatic hydrolysis included neutral protease, and the enzymes used in the second enzymatic hydrolysis included alkaline protease and papain.

4. The preparation method according to claim 3, characterized in that, The temperature of the first enzymatic hydrolysis is 35-40℃, and the time of the first enzymatic hydrolysis is 55-65 min; the amount of neutral protease added is 0.018%-0.022% of the mass of Spirulina.

5. The preparation method according to claim 3, characterized in that, The first enzymatic hydrolysis is followed by homogenization, which is performed at a pressure of 33-38 kPa and is repeated 1-2 times.

6. The preparation method according to claim 3, characterized in that, The enzyme used in the second enzymatic hydrolysis is added in several stages, including an initial addition and a final addition; the time interval between the initial and final additions is ≤30 minutes. The enzymes added for the first time include alkaline protease and papain; The enzyme added in the last addition includes alkaline protease; the enzymatic hydrolysis time is started after the last addition, and the enzymatic hydrolysis time is 4-5 hours.

7. The preparation method according to claim 6, characterized in that, The mass of the enzyme added for the first time is 0.23% to 0.27% of the mass of spirulina; the mass of the enzyme added for the last time is 0.07% to 0.09% of the mass of spirulina.

8. The preparation method according to claim 2 or 5, characterized in that, The second enzymatic hydrolysis is performed at a temperature of 50–55°C and a pH of 8.0–8.5; the enzymatic hydrolysis is followed by purification and drying.

9. The use of the spirulina peptide according to claim 1 or the spirulina peptide prepared by any one of claims 2 to 8 in the preparation of products for antiviral pneumonia.

10. A product for treating viral pneumonia, characterized in that, This includes the spirulina peptide according to claim 1 or the spirulina peptide prepared by the preparation method according to any one of claims 2 to 8.

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