A blood protein-specific active peptide with the functions of replenishing qi and blood, improving microcirculation and promoting vasodilation, its preparation method and application

Abstract

This application discloses a precise active peptide of hemoglobin with the functions of replenishing qi and blood, improving microcirculation, and promoting vasodilation, as well as its preparation method and application, belonging to the field of hemoglobin peptide technology. The precise active peptide of hemoglobin contains at least one of LGFP, IGFP, FPHFN, LFL, LFI, FESF, ILF, LIF, FLL, FIL, FII, or FPHFD. The prepared precise active peptide of hemoglobin exhibits multiple biological activities: it exerts a hypotensive effect by inhibiting ACE and vasoconstrictive substances; it promotes vasodilation by activating the PI3K / AKT / eNOS signaling pathway; and it has been shown to significantly improve microcirculation and replenish qi and blood in a zebrafish model. The preparation method has low equipment requirements, is simple and easy to operate, and is suitable for large-scale industrial production. This invention enriches the bioactive peptide library and is of great significance for promoting technological progress and industrial upgrading in the field of hemoglobin peptides.

Description

Technical Field

[0001] This invention relates to the field of hemoglobin peptide technology, specifically to a hemoglobin-specific active peptide that has the functions of replenishing qi and blood, improving microcirculation and promoting vasodilation, as well as its preparation method and application. Background Technology

[0002] Hypertension and endothelial dysfunction are major contributing factors to cardiovascular disease. Their core pathological mechanisms include decreased nitric oxide (NO) bioavailability, excessive endothelin-1 (ET-1) secretion, and oxidative stress damage. While commonly used vasodilators (such as calcium channel blockers and NO donor drugs) have some efficacy, they suffer from significant side effects (such as hypotension and headache) and poor long-term tolerability. Therefore, developing natural, safe, and multi-target active ingredients that can improve vascular function has become a research hotspot.

[0003] Food-derived bioactive peptides have attracted much attention due to their high safety and multiple regulatory functions. Pig blood, as a byproduct of meat processing, is an ideal raw material for preparing vasoactive peptides because it is rich in high-quality protein. Existing studies have shown that pig blood enzymatic hydrolysates contain various short peptides with ACE inhibitory activity (such as Val-Pro-Pro and Ile-Pro-Pro), but research on their direct regulatory effects on vascular endothelial function is limited. In particular, the characteristic peptides that can simultaneously promote eNOS / NO pathway activation and inhibit ET-1 secretion remain unclear. Currently reported vasodilatory peptides are mostly derived from milk proteins (such as Casokinins derived from K-casein) or fish collagen (such as Gly-Pro-Hyp), while specific peptides with similar functions in pig blood proteins are still largely unexplored. Peptides obtained by traditional enzymatic hydrolysis processes typically have a wide molecular weight distribution, unstable activity, and lack targeting specificity to vascular endothelial cells. Furthermore, existing patents mainly focus on the hypotensive effects of ACE inhibitory peptides, with insufficient coverage of the direct regulatory mechanisms of endothelium-dependent vasodilation.

[0004] With the deepening research on bioactive peptides, peptides have shown great potential in improving human health due to their high bioactivity, low toxicity, and good targeting properties. In traditional Chinese medicine's "Qi and Blood Theory," abundant Qi and Blood and smooth circulation are key to maintaining bodily health. Modern medical research shows that certain bioactive peptides can exert physiological effects similar to "replenishing Qi and Blood" through mechanisms such as promoting hematopoietic function and improving microcirculation. Existing hemoprotein peptide products mostly focus on their nutritional supplementation function, while insufficient exploration of their specific effects in hematopoietic regulation and microcirculation improvement. Research on the structure-activity relationship of hemoprotein peptides is still inadequate, and there is a lack of precise peptide design targeting specific functions.

[0005] Therefore, identifying specific fragments of hemorrhagic protein peptides that have effects on vasodilation and replenishing qi and blood has important theoretical value and practical significance. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a blood protein precise active peptide that can replenish qi and blood, improve microcirculation and promote vasodilation, as well as its preparation method and application.

[0007] According to a first aspect of the present invention, a blood protein precise active peptide with functions of replenishing qi and blood, improving microcirculation and promoting vasodilation is provided, wherein the blood protein precise active peptide comprises at least one of LGFP, IGFP, FPHFN, LFL, LFI, FESF, ILF, LIF, FLL, FIL, FII or FPHFD.

[0008] The specific sequences of LGFP are shown in SEQ ID NO.1; IGFP is shown in SEQ ID NO.2; FPHFN is shown in SEQ ID NO.3; FESF is shown in SEQ ID NO.4; and FPHFD is shown in SEQ ID NO.5.

[0009] Furthermore, the blood protein-specific active peptide comprises at least one of LGFP, IGFP, LFL, LFI, or FESF.

[0010] According to a second aspect of the present invention, a method for preparing a blood protein-specific active peptide with functions of replenishing qi and blood, improving microcirculation, and promoting vasodilation is provided, comprising the following steps:

[0011] S1. Mix hemoglobin powder with water evenly and homogenize to obtain a pretreatment solution;

[0012] S2. Add Bacillus subtilis neutral protease to the pretreatment solution for the first hydrolysis and enzyme inactivation to obtain the first hydrolysate.

[0013] S3. Add acidic protease to the first hydrolysate for a second hydrolysis and enzyme inactivation to obtain a second hydrolysate;

[0014] S4. The second hydrolysate is filtered, ultrafiltered and spray-dried to obtain blood protein precise active peptides;

[0015] The prepared blood protein precise active peptide contains at least one of LGFP, IGFP, FPHFN, LFL, LFI, FESF, ILF, LIF, FLL, FIL, FII, or FPHFD;

[0016] The specific sequences of LGFP are shown in SEQ ID NO.1; IGFP is shown in SEQ ID NO.2; FPHFN is shown in SEQ ID NO.3; FESF is shown in SEQ ID NO.4; and FPHFD is shown in SEQ ID NO.5.

[0017] Furthermore, in S1, the mass ratio of hemoglobin powder to water is 1:(5~15); the homogenization treatment time is 30~90 min.

[0018] Furthermore, the amount of neutral protease from Bacillus subtilis added is 3% to 4% of the mass of the hemoglobin powder; the amount of acidic protease added is 0.2% to 0.8% of the mass of the hemoglobin powder.

[0019] Furthermore, the neutral protease of Bacillus subtilis has an enzyme activity of 80,000 U / g, and the acidic protease has an enzyme activity of 600,000 U / g.

[0020] Furthermore, the temperature of the first hydrolysis is 50℃~55℃, the pH of the hydrolysis reaction is 6.5~7.0, and the hydrolysis time is 2~4h; the temperature of the second hydrolysis is 35℃~40℃, the pH of the hydrolysis reaction is 2.0~3.0, and the hydrolysis time is 2~4h.

[0021] Furthermore, the enzyme inactivation conditions described in S2 and S3 are both at 85℃~90℃ for 15~20min, the filtration method described in S4 is solid-liquid separation, and the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 5000 Da.

[0022] Furthermore, the blood protein-specific active peptide comprises at least one of LGFP, IGFP, LFL, LFI, or FESF.

[0023] According to a third aspect of the present invention, the application of the blood protein precise active peptide as described herein in the preparation of functional products for replenishing qi and blood, improving microcirculation, or promoting vasodilation is proposed.

[0024] According to a fourth aspect of the present invention, the application of a blood protein-specific active peptide prepared by the method described herein in the preparation of functional products for replenishing qi and blood, improving microcirculation, or promoting vasodilation is proposed.

[0025] The beneficial effects of this invention are:

[0026] The blood protein-specific active peptides prepared by the method of this invention exhibit ACE inhibition, blood pressure reduction, inhibition of vasoconstrictor production, and vasodilation promotion in endothelial cells via the PI3K / AKT / eNOS pathway. In zebrafish, they improve microcirculation by increasing blood flow velocity and replenish qi and blood. Furthermore, the blood protein-specific active peptides prepared by the method of this invention have no obvious bitter or astringent taste. Moreover, the preparation method of this invention requires minimal equipment, is simple and easy to operate, and is suitable for large-scale industrial production. The desired blood protein-specific active peptides can be obtained through a simple combination of operations such as mixing, pretreatment, enzymatic hydrolysis, and filtration. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention.

[0028] Figure 1 This is a cell viability diagram of the blood protein precise active peptide after being treated with H2O2, according to a specific embodiment of the present invention.

[0029] Figure 2 This is a graph showing the effect of the blood protein-specific active peptide on the expression level of eNOS mRNA in HUVEC cells according to a specific embodiment of the present invention.

[0030] Figure 3 This is a graph showing the effect of the blood protein-specific active peptide on the expression level of AKT1 mRNA in HUVEC cells according to a specific embodiment of the present invention.

[0031] Figure 4 This is a graph showing the effect of the blood protein-specific active peptide on the expression level of PI3K mRNA in HUVEC cells according to a specific embodiment of the present invention.

[0032] Figure 5 This is a graph showing the effect of the blood protein-specific active peptide on NOS3 content in an endothelial dysfunction model according to a specific embodiment of the present invention.

[0033] Figure 6 This is a graph showing the effect of the blood protein-specific active peptide on the ET-1 content in an endothelial dysfunction model according to a specific embodiment of the present invention.

[0034] Figure 7 This is a typical image showing the staining intensity of erythrocytes in the heart of zebrafish after sample processing, according to a specific embodiment of the present invention.

[0035] Figure 8 This is a staining intensity diagram of zebrafish heart erythrocytes after sample treatment, according to a specific embodiment of the present invention.

[0036] Figure 9 This is a blood flow velocity diagram of zebrafish after sample treatment, according to a specific embodiment of the present invention.

[0037] Figure 10 This is a 2D diagram of the docking of LGFP and eNOS molecules in a specific embodiment of the present invention;

[0038] Figure 11 This is a 2D diagram of the docking of IGFP and eNOS molecules in a specific embodiment of the present invention;

[0039] Figure 12 This is a 2D diagram of the docking of LFL and eNOS molecules in a specific embodiment of the present invention;

[0040] Figure 13 This is a 2D diagram of the docking of LFI and eNOS molecules in a specific embodiment of the present invention;

[0041] Figure 14 This is a 2D diagram of the docking of FESF and eNOS molecules in a specific embodiment of the present invention;

[0042] Figure 15 This is a 2D diagram of the docking of LGFP and AKT1 molecules in a specific embodiment of the present invention;

[0043] Figure 16 This is a 2D diagram of the docking of IGFP and AKT1 molecules in a specific embodiment of the present invention;

[0044] Figure 17 This is a 2D diagram of the docking of LFL and AKT1 molecules in a specific embodiment of the present invention;

[0045] Figure 18 This is a 2D diagram of the docking of LFI and AKT1 molecules in a specific embodiment of the present invention;

[0046] Figure 19 This is a 2D diagram of the docking of FESF and AKT1 molecules in a specific embodiment of the present invention;

[0047] Figure 20 This is a 2D diagram of the docking of LGFP and PI3K molecules in a specific embodiment of the present invention;

[0048] Figure 21 This is a 2D diagram of the docking of IGFP and PI3K molecules in a specific embodiment of the present invention;

[0049] Figure 22 This is a 2D diagram of the docking of LFL and PI3K molecules in a specific embodiment of the present invention;

[0050] Figure 23 This is a 2D diagram of the docking of LFI and PI3K molecules in a specific embodiment of the present invention;

[0051] Figure 24 This is a 2D diagram of the docking of FESF and PI3K molecules in a specific embodiment of the present invention;

[0052] Figure 25 This is a 2D diagram of the docking of LGFP with the ETA receptor molecule in a specific embodiment of the present invention;

[0053] Figure 26 This is a 2D diagram of the docking of IGFP with the ETA receptor molecule in a specific embodiment of the present invention;

[0054] Figure 27 This is a 2D diagram of the docking of LFL with the ETA receptor molecule in a specific embodiment of the present invention;

[0055] Figure 28 This is a 2D diagram of the docking of LFI with the ETA receptor molecule in a specific embodiment of the present invention;

[0056] Figure 29 This is a 2D diagram of the docking of FESF with the ETA receptor molecule in a specific embodiment of the present invention;

[0057] Figure 30 This is a 2D diagram of the docking of LGFP with the G-CSF receptor molecule in a specific embodiment of the present invention;

[0058] Figure 31 This is a 2D diagram of the docking of IGFP with the G-CSF receptor molecule in a specific embodiment of the present invention;

[0059] Figure 32 This is a 2D diagram of the docking of LFL with the G-CSF receptor molecule in a specific embodiment of the present invention;

[0060] Figure 33 This is a 2D diagram of the docking of LFI with the G-CSF receptor molecule in a specific embodiment of the present invention;

[0061] Figure 34 This is a 2D diagram of the docking of FESF and G-CSF receptor molecules in a specific embodiment of the present invention;

[0062] Figure 35 This is a 2D diagram of the docking of LGFP with the GATA1 receptor molecule in a specific embodiment of the present invention;

[0063] Figure 36 This is a 2D diagram of the docking of IGFP with the GATA1 receptor molecule in a specific embodiment of the present invention;

[0064] Figure 37 This is a 2D diagram of the docking of LFL with the GATA1 receptor molecule in a specific embodiment of the present invention;

[0065] Figure 38 This is a 2D diagram of the docking of LFI with the GATA1 receptor molecule in a specific embodiment of the present invention;

[0066] Figure 39 This is a 2D diagram of the docking of FESF with the GATA1 receptor molecule in a specific embodiment of the present invention;

[0067] Reference numbers: 1-GLY B: 186; 2-TRP B: 356; 3-CYS B: 184; 4-VAL B: 336; 5-PRO B: 334; 6-SER B: 354; 7-GLU B: 361; 8-GLY B: 355; 9-PHE B: 353; 10-TYR B: 475; 11-ARG 18-ILE A:1150;22-ALA A:1130;23-LEU A:101; 24-GLY A:58; 25-ASN B:30; 26-ILE A:57; 27-VAL A:59; 28-VAL B:59; 29-GLY B:58; 30-ILE A:37; 31-ALA B:33; 32-ILE B:37; 33-ASP B:34. Detailed Implementation

[0068] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0069] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0070] The first objective of this invention is to propose a blood protein precise active peptide that has the functions of replenishing qi and blood, improving microcirculation and promoting vasodilation, wherein the blood protein precise active peptide has no obvious bitter or astringent taste.

[0071] The second objective of this invention is to provide a method for preparing a blood protein-specific active peptide with functions of replenishing qi and blood, improving microcirculation, and promoting vasodilation. The preparation method has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production. The desired blood protein-specific active peptide can be obtained by simply combining operations such as mixing, pretreatment, enzymatic hydrolysis, and filtration.

[0072] The third objective of this invention is to propose the application of the blood protein precise active peptide as described above in the preparation of functional products for replenishing qi and blood, improving microcirculation, or promoting vasodilation.

[0073] The fourth objective of this invention is to propose the application of a blood protein-specific active peptide prepared by the method described above in the preparation of functional products for replenishing qi and blood, improving microcirculation, or promoting vasodilation.

[0074] To achieve the above objectives, the present invention provides a blood protein precise active peptide that has the functions of replenishing qi and blood, improving microcirculation and promoting vasodilation, characterized in that the blood protein precise active peptide contains at least one of LGFP, IGFP, FPHFN, LFL, LFI, FESF, ILF, LIF, FLL, FIL, FII or FPHFD.

[0075] The specific sequences of LGFP are shown in SEQ ID NO.1; IGFP is shown in SEQ ID NO.2; FPHFN is shown in SEQ ID NO.3; FESF is shown in SEQ ID NO.4; and FPHFD is shown in SEQ ID NO.5. The blood protein-specific active peptide comprises at least one of LGFP, IGFP, LFL, LFI, or FESF.

[0076] This invention also provides a method for preparing a blood protein-specific active peptide that replenishes qi and blood, improves microcirculation, and promotes vasodilation, specifically including the following steps:

[0077] (1) Mix hemoglobin powder with water evenly, wherein the mass ratio of hemoglobin powder to pure water is 1: (5-15), homogenize for 30-90 min to obtain a pretreatment solution.

[0078] (2) Add Bacillus subtilis neutral protease (enzyme activity 80,000 U / g) to the pretreatment solution, adjust the pH of the system to 6.5-7.0, hydrolyze at 50℃-55℃ for 2-4 h, and then inactivate the enzyme at 85℃-90℃ for 15-20 min to obtain the first hydrolysate; wherein, the amount of Bacillus subtilis neutral protease added is 3%-4% of the mass of hemoglobin powder;

[0079] (3) Add acidic protease (enzyme activity 600,000 U / g) to the first hydrolysate, adjust the pH of the system to 2.0-3.0, hydrolyze at 35℃-40℃ for 2-4 hours, and then inactivate the enzyme at 85℃-90℃ for 15-20 minutes to obtain the second hydrolysate; wherein, the amount of acidic protease added is 0.2%-0.8% of the mass of hemoglobin powder.

[0080] (4) The third hydrolysate is cooled to room temperature and filtered. After filtration, the filtrate with a molecular weight of less than 5000 Da is obtained by ultrafiltration and then spray-dried to obtain the blood protein precise active peptide.

[0081] (5) The activity of blood protein precise active peptides was detected and cell experiments were conducted. It has ACE inhibition and blood pressure reduction effects, can inhibit the generation of vasoconstrictive substances, and can exert its vasodilatory effect in endothelial cells through the PI3K / AKT / eNOS pathway. In zebrafish, it can also improve microcirculation by increasing blood flow velocity, and also has the effects of replenishing qi and blood.

[0082] The enzymatic hydrolysis process of this invention is characterized by hydrolyzing hemoglobin with Bacillus subtilis neutral protease to obtain polypeptides whose C-terminus is mainly composed of hydrophobic amino acids (Leu, Ile, Val) and small molecule amino acids (Ala, Gly); and further hydrolyzing with acidic protease to obtain polypeptides whose C-terminus is mainly composed of aromatic amino acids and leucine, thereby greatly increasing the content of core peptides.

[0083] It should be noted that the enzymes with the same name used in the following examples and comparative examples are from the same commercially available company and brand, meaning that the enzyme activities of the enzymes with the same name used in each example and comparative example are consistent. The Bacillus subtilis neutral protease with an activity of 80,000 U / g, the acidic protease with an activity of 600,000 U / g, the Bacillus licheniformis alkaline protease with an activity of 400,000 U / g, and the trypsin with an activity of 250,000 U / g used in the examples and comparative examples.

[0084] Example 1

[0085] 1. Take 100g of hemoglobin powder (protein content 96%), mix it with 1000g of pure water, and homogenize for 60 minutes;

[0086] 2. Heat to 55℃, adjust pH to 7.0 with sodium hydroxide, add 3.45g of Bacillus subtilis neutral protease for 3h hydrolysis, and heat to 85℃ for 20min to inactivate enzyme;

[0087] 3. After cooling to 37℃, adjust the pH to 2.5 with hydrochloric acid, add 0.5g of acidic protease to hydrolyze for 3h, and heat to 85℃ to inactivate the enzyme for 20min;

[0088] 4. After filtration, ultrafiltration is performed to obtain a blood protein precise active peptide solution with a molecular weight of less than 5000 Da.

[0089] 5. Blood protein precise active peptide powder is obtained by spray drying.

[0090] Example 2

[0091] 1. Take 100g of hemoglobin powder (protein content 96%), mix it with 500g of pure water, and homogenize for 90 minutes;

[0092] 2. Heat to 55℃, adjust pH to 6.5 with sodium hydroxide, add 4g of Bacillus subtilis neutral protease to hydrolyze for 2h, and heat to 90℃ to inactivate enzyme for 15min;

[0093] 3. After cooling to 35℃, adjust the pH to 2.5 with hydrochloric acid, add 0.2g of acidic protease to hydrolyze for 4h, and heat to 85℃ to inactivate the enzyme for 20min;

[0094] 4. After filtration, ultrafiltration is performed to obtain a blood protein precise active peptide solution with a molecular weight of less than 5000 Da.

[0095] 5. Blood protein precise active peptide powder is obtained by spray drying.

[0096] Example 3

[0097] 1. Take 100g of hemoglobin powder (protein content 96%), mix it with 1500g of pure water, and homogenize for 30 minutes;

[0098] 2. Heat to 55℃, adjust pH to 6.8 with sodium hydroxide, add 3g of Bacillus subtilis neutral protease to hydrolyze for 4h, and heat to 90℃ to inactivate enzyme for 20min;

[0099] 3. After cooling to 40℃, adjust the pH to 3.0 with hydrochloric acid, add 0.8g of acidic protease to hydrolyze for 2h, and heat to 90℃ to inactivate the enzyme for 20min;

[0100] 4. After filtration, ultrafiltration is performed to obtain a blood protein precise active peptide solution with a molecular weight of less than 5000 Da.

[0101] 5. Blood protein precise active peptide powder is obtained by spray drying.

[0102] Comparative Example 1

[0103] The hydrolysis process in this comparative example uses only Bacillus subtilis alkaline protease. The specific experimental steps differ from those in Example 1 only in that: the Bacillus subtilis neutral protease in step 2 is replaced with Bacillus subtilis alkaline protease, the amount added is 1.44g, the pH is 8.0, and step 3 is removed. The operation and process of the remaining experimental steps are the same as those in Example 1.

[0104] In Example 1, the amount of Bacillus subtilis neutral protease added was 3.45g. Based on the protein content (96%) in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g. The specific calculation process is as follows:

[0105] The enzyme activity of neutral protease in 3.45g of Bacillus subtilis was 80,000 U / g. 3.45g = 276,000 U, which translates to an enzyme addition of 276,000 U / (100g) based on protein content per gram of hemoglobin powder. 96%)g=2875U / g (the denominator g in the unit represents the amount of protein in each gram of hemoglobin powder); similarly, the amount of acidic protease added is 0.5g, which, based on the amount of protein in the hemoglobin powder, is 3125U / g.

[0106] Therefore, in this comparative example, the amount of Bacillus licheniformis alkaline protease added was 1.44g, which, based on the protein content in the hemoglobin powder, is 6000 U / g of Bacillus licheniformis alkaline protease added; this is the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0107] Comparative Example 2

[0108] The hydrolysis process in this comparative example uses only Bacillus subtilis neutral protease. The specific experimental steps differ from those in Example 1 only in that the amount of Bacillus subtilis neutral protease added in step 2 is changed to 7.2g, and step 3 is removed. The operation and process of the remaining experimental steps are the same as those in Example 1.

[0109] In Example 1, the amount of Bacillus subtilis neutral protease added was 3.45g. Based on the protein content (96%) in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g. The specific calculation process is as follows:

[0110] The enzyme activity of neutral protease in 3.45g of Bacillus subtilis was 80,000 U / g. 3.45g = 276,000 U, which translates to an enzyme addition of 276,000 U / (100g) based on protein content per gram of hemoglobin powder. 96%)g=2875U / g (the denominator g in the unit represents the amount of protein in each gram of hemoglobin powder); similarly, the amount of acidic protease added is 0.5g, which, based on the amount of protein in the hemoglobin powder, is 3125U / g.

[0111] Therefore, the 7.2g of Bacillus subtilis neutral protease in this comparative example, based on the protein content in the hemoglobin powder, is 6000 U / g, which is the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0112] Comparative Example 3

[0113] The hydrolysis process in this comparative example uses only a single acidic protease. The only difference between the specific experimental steps and those in Example 1 is that the amount of acidic protease added in step 3 is changed to 0.96g, and the operation and process of the remaining experimental steps are the same as those in Example 1.

[0114] In Example 1, the amount of Bacillus subtilis neutral protease added was 3.45g. Based on the protein content (96%) in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g. The specific calculation process is as follows:

[0115] The enzyme activity of neutral protease in 3.45g of Bacillus subtilis was 80,000 U / g. 3.45g = 276,000 U, which translates to an enzyme addition of 276,000 U / (100g) based on protein content per gram of hemoglobin powder. 96%)g=2875U / g (the denominator g in the unit represents the amount of protein in each gram of hemoglobin powder); similarly, the amount of acidic protease added is 0.5g, which, based on the amount of protein in the hemoglobin powder, is 3125U / g.

[0116] Therefore, the 0.96g acidic protease in this comparative example, based on the protein content in the hemoglobin powder, is 6000 U / g, which is the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0117] Comparative Example 4

[0118] The hydrolysis process in this comparative example uses only trypsin. The specific experimental steps differ from those in Example 1 in that: the Bacillus subtilis neutral protease in step 2 of Example 1 is replaced with trypsin, the amount added is changed to 2.304g, step 3 is removed, and the operation and process of the remaining experimental steps are the same as in Example 1.

[0119] In Example 1, the amount of Bacillus subtilis neutral protease added was 3.45g. Based on the protein content (96%) in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g. The specific calculation process is as follows:

[0120] The enzyme activity of neutral protease in 3.45g of Bacillus subtilis was 80,000 U / g. 3.45g = 276,000 U, which translates to an enzyme addition of 276,000 U / (100g) based on protein content per gram of hemoglobin powder. 96%)g=2875U / g (the denominator g in the unit represents the amount of protein in each gram of hemoglobin powder); similarly, the amount of acidic protease added is 0.5g, which, based on the amount of protein in the hemoglobin powder, is 3125U / g.

[0121] Therefore, the amount of Bacillus subtilis neutral protease added to the 2.304g of Bacillus subtilis neutral protease in this comparative example, based on the protein content in the hemoglobin powder, is 6000 U / g, which is the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0122] Comparative Example 5

[0123] The hydrolysis process of this comparative example uses Bacillus subtilis neutral protease and Bacillus licheniformis alkaline protease. The specific experimental steps are different from those of Example 1 in that the acidic protease in step 3 of Example 1 is replaced with Bacillus licheniformis alkaline protease, the amount added is changed to 0.75g, and the enzymatic hydrolysis pH is 8.0. The operation and process of the remaining experimental steps are the same as those of Example 1.

[0124] In Example 1, the amount of Bacillus subtilis neutral protease added was 3.45g. Based on the protein content (96%) in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g. The specific calculation process is as follows:

[0125] The enzyme activity of neutral protease in 3.45g of Bacillus subtilis was 80,000 U / g. 3.45g = 276,000 U, which translates to an enzyme addition of 276,000 U / (100g) based on protein content per gram of hemoglobin powder. 96%)g=2875U / g (the denominator g in the unit represents the amount of protein in each gram of hemoglobin powder); similarly, the amount of acidic protease added is 0.5g, which, based on the amount of protein in the hemoglobin powder, is 3125U / g.

[0126] Therefore, the amount of Bacillus subtilis neutral protease added in this comparative example, based on the protein content in the hemoglobin powder, is 2875 U / g, and the amount of Bacillus licheniformis alkaline protease added, based on the protein content in the hemoglobin powder, is 3125 U / g, which is the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0127] Comparative Example 6

[0128] The hydrolysis process in this comparative example only uses Bacillus subtilis alkaline protease and acidic protease. The specific experimental steps differ from those in Example 1 in that: the Bacillus subtilis neutral protease in step 2 is replaced with Bacillus subtilis alkaline protease, the amount added is changed to 0.69g, and the pH is 8.0; the operation and process of the remaining experimental steps are the same as in Example 1.

[0129] In Example 1, the amount of Bacillus subtilis neutral protease added was 3.45g. Based on the protein content (96%) in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g. The specific calculation process is as follows:

[0130] The enzyme activity of neutral protease in 3.45g of Bacillus subtilis was 80,000 U / g. 3.45g = 276,000 U, which translates to an enzyme addition of 276,000 U / (100g) based on protein content per gram of hemoglobin powder. 96%)g=2875U / g (the denominator g in the unit represents the amount of protein in each gram of hemoglobin powder); similarly, the amount of acidic protease added is 0.5g, which, based on the amount of protein in the hemoglobin powder, is 3125U / g.

[0131] Therefore, the amount of Bacillus licheniformis alkaline protease added in this comparative example, based on the protein content in the hemoglobin powder, is 2875 U / g, and the amount of Bacillus licheniformis acidic protease added, based on the protein content in the hemoglobin powder, is 3125 U / g, which is the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0132] Comparative Example 7

[0133] The hydrolysis process in this comparative example only uses Bacillus subtilis alkaline protease and trypsin. The specific experimental steps differ from those in Example 1 in that: in step 2, Bacillus subtilis neutral protease is replaced with Bacillus subtilis alkaline protease, the amount added is changed to 1g, and the pH is 8.0; in step 3, acidic protease is replaced with trypsin, the amount added is changed to 0.704g, the enzymatic hydrolysis temperature is 55℃, and the pH is 7.0; the operation and process of the remaining experimental steps are the same as in Example 1.

[0134] In Example 1, the amount of Bacillus subtilis neutral protease added was 3.45g. Based on the protein content (96%) in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g. The specific calculation process is as follows:

[0135] The enzyme activity of neutral protease in 3.45g of Bacillus subtilis was 80,000 U / g. 3.45g = 276,000 U, which translates to an enzyme addition of 276,000 U / (100g) based on protein content per gram of hemoglobin powder. 96%)g=2875U / g (the denominator g in the unit represents the amount of protein in each gram of hemoglobin powder); similarly, the amount of acidic protease added is 0.5g, which, based on the amount of protein in the hemoglobin powder, is 3125U / g.

[0136] Therefore, the amount of Bacillus licheniformis alkaline protease added in this comparative example, based on the protein content in the hemoglobin powder, is 4167 U / g, and the amount of trypsin added in 0.74g, based on the protein content in the hemoglobin powder, is 1833 U / g, which is the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0137] Comparative Example 8

[0138] The hydrolysis process of this comparative example uses Bacillus subtilis neutral protease and trypsin. The specific experimental steps differ from those of Example 1 in that the acidic protease in step 3 is replaced with trypsin, the amount added is changed to 1.2g, and the pH is 7.0; the operation and process of the remaining experimental steps are the same as those of Example 1.

[0139] In Example 1, the amount of Bacillus subtilis neutral protease added was 3.45g. Based on the protein content (96%) in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g. The specific calculation process is as follows:

[0140] The enzyme activity of neutral protease in 3.45g of Bacillus subtilis was 80,000 U / g. 3.45g = 276,000 U, which translates to an enzyme addition of 276,000 U / (100g) based on protein content per gram of hemoglobin powder. 96%)g=2875U / g (the denominator g in the unit represents the amount of protein in each gram of hemoglobin powder); similarly, the amount of acidic protease added is 0.5g, which, based on the amount of protein in the hemoglobin powder, is 3125U / g.

[0141] Therefore, the amount of Bacillus licheniformis alkaline protease added in this comparative example, based on the protein content in the hemoglobin powder, is 2875 U / g, and the amount of trypsin added, based on the protein content in the hemoglobin powder, is 3125 U / g, which is the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0142] Comparative Example 9

[0143] The hydrolysis process in this comparative example uses trypsin and acidic protease. The specific experimental steps differ from those in Example 1 in that: the Bacillus subtilis neutral protease in step 2 is replaced with trypsin, the amount added is changed to 1.104g, and the pH is 7.0; the operation and process of the remaining experimental steps are the same as in Example 1.

[0144] In Example 1, the amount of Bacillus subtilis neutral protease added was 3.45g. Based on the protein content (96%) in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g. The specific calculation process is as follows:

[0145] The enzyme activity of neutral protease in 3.45g of Bacillus subtilis was 80,000 U / g. 3.45g = 276,000 U, which translates to an enzyme addition of 276,000 U / (100g) based on protein content per gram of hemoglobin powder. 96%)g=2875U / g (the denominator g in the unit represents the amount of protein in each gram of hemoglobin powder); similarly, the amount of acidic protease added is 0.5g, which, based on the amount of protein in the hemoglobin powder, is 3125U / g.

[0146] Therefore, the 1.104g trypsin in this comparative example, based on the protein content in the hemoglobin powder, has an enzyme addition of 2875 U / g, and the 0.5g acidic protease, based on the protein content in the hemoglobin powder, has an enzyme addition of 3125 U / g, which is the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0147] Comparative Example 10

[0148] The hydrolysis process in this comparative example uses acidic protease and neutral protease. The specific experimental steps differ from those in Example 1 in that the order of steps 2 and 3 is reversed, i.e., acidic protease is used for hydrolysis first, followed by neutral protease. The operation and process of the remaining experimental steps are the same as in Example 1.

[0149] The amount of enzyme preparation added was the same as in Example 1, namely, in step 2, the pH was adjusted to 2.5, and 0.5g of acidic protease was added (based on the protein content in the hemoglobin powder, the amount of acidic protease added was 3125 U / g); in step 2, the pH was adjusted to 7.0, and 3.45g of Bacillus subtilis neutral protease was added (based on the protein content in the hemoglobin powder, the amount of Bacillus subtilis neutral protease added was 2875 U / g). This was the same as the total amount of Bacillus subtilis neutral protease and acidic protease used in Example 1.

[0150] Example 4

[0151] This embodiment describes the activity detection, cell and animal experiments conducted on the blood protein precise active peptide products prepared in Examples 1 to 3 and Comparative Examples 1 to 10.

[0152] 1. VVYP content of blood protein precise active peptides

[0153] Patent CN 101622945 A describes how a special health food containing a certain amount of Val-Val-Tyr-Pro (VVYP) peptide can effectively prevent or improve symptoms such as tension, anxiety, and low stress response. Therefore, the VVYP content in the examples and comparative examples was tested.

[0154] 1.1 Sample Pretreatment

[0155] A: Preparation of the mobile phase:

[0156] Mobile phase A: A pure aqueous solution containing 0.1% trifluoroacetic acid

[0157] Mobile phase B: Pure acetonitrile solution containing 0.1% trifluoroacetic acid

[0158] B: Sample preparation: Accurately pipette 500 mg of sample into a 10 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, take 1 mL and filter through a microporous membrane (0.22 μm, aqueous system) for later use.

[0159] C: Standard: Accurately pipette 1.0 mg (accurate to 0.01 mg) of VVYP standard into a 10 mL volumetric flask, dissolve in water and dilute to the mark, shake well, take 1 mL and filter through a microporous membrane (0.22 μm, aqueous system) for later use.

[0160] 1.2 Detection Method

[0161] High-performance liquid chromatography (HPLC)

[0162] Chromatographic conditions:

[0163] Chromatographic column: 150 mm × 4.6 mm, 3 μm, ChromCore C18 column.

[0164] Mobile phase:

[0165] Phase A: 0.1% trifluoroacetic acid (TFA) aqueous solution.

[0166] Phase B: 0.1% TFA acetonitrile solution.

[0167] The elution gradient is shown in Table 1:

[0168] Table 1 Elution gradient

[0169]

[0170] Washing method: isocratic washing

[0171] Flow rate: 1.0 mL / min.

[0172] Detection wavelength: 220 nm (peptide bond absorption wavelength).

[0173] Quantitative analysis: A standard curve was plotted using VVYP standards, and the content of VVYP in the sample was calculated based on the peak area, as shown in formula (1).

[0174] VVYP content (%) = [(F2×C×10) / (F1×m×10)] ×100% (1)

[0175] F1 is the standard peak volume.

[0176] F2 is the peak area of ​​the sample.

[0177] C is the mass concentration of the standard, in mg / mL.

[0178] m is the sample mass, g

[0179] 10 is the final volume of the sample, in mL.

[0180] 1000 is the unit conversion factor.

[0181] 1.3 Results and Analysis

[0182] Table 2. VVYP content in samples

[0183]

[0184] As can be seen from the VVYP content results in Table 2, the VVYP content of Examples 1, 2, 3 and Comparative Examples 3, 4, 8, 9 is all above 0.4%, which is significantly different from the other comparative examples. Therefore, Examples 1, 2, 3 and Comparative Examples 3, 4, 8, 9 were selected for the next step of the experiment.

[0185] 2. Inhibitory activity of blood protein-specific active peptides

[0186] Based on the results of VVYP, Examples 1, 2, and 3 and Comparative Examples 3, 4, 8, and 9 were selected for ACE inhibition activity testing.

[0187] 2.1 Experimental Methods

[0188] ACE (angiotensin-converting enzyme) hydrolyzes the substrate hippuryl-histidyl-leucine (HHL) to produce hippuric acid (HA). The inhibitory activity of ACE can be assessed by detecting the amount of hippuric acid produced.

[0189] Add 50 μL of ACE enzyme solution, 50 μL of the test sample, and 150 μL of HHL substrate solution (dissolved in borate buffer) to a test tube. Mix well and incubate at 37°C for 30 minutes. Add 250 μL of 1 M HCl to terminate the reaction. Add 1.5 mL of ethyl acetate, vortex to mix, and centrifuge (3000×g, 10 minutes). Take the upper organic phase, dry it, and reconstitute it with 1 mL of distilled water. Measure the absorbance at 228 nm using a UV spectrophotometer. Calculate the amount of hippuric acid produced in the sample according to the hippuric acid standard curve, as shown in formula (2).

[0190] ACE inhibition rate (%) = [(control group absorbance - sample group absorbance) / control group absorbance] × 100% (2)

[0191] 2.2 Experimental Results and Analysis

[0192] Table 3 ACE inhibitory activity

[0193]

[0194] It can be seen that Examples 1, 2, 3 and Comparative Examples 3, 4, 8, 9 all have certain ACE inhibitory activities, indicating that they all have certain antihypertensive effects. Moreover, the ACE inhibitory activities of each group increase with the increase of concentration. Among them, the ACE inhibitory activity of Example 1 reached 68.64% at 1 mg / mL.

[0195] 3. The effect of blood protein-specific active peptides on improving vascular endothelial dysfunction.

[0196] Cell viability was determined using the CCK8 assay (Cell Counting Kit-8). The results were analyzed for significance, and the highest non-toxic concentration of 1 mg / mL was selected for drug administration to the cells.

[0197] 3.1 Establishing an endothelial dysfunction model by inducing H2O2-induced damage to HUVEC cells

[0198] HUVEC cells were cultured with different concentrations of H2O2 (0.2mM, 0.4mM, 0.6mM, 0.8mM and 1mM) for 1 h. Cell viability was measured by adding CCK8 reagent. At 0.6mM, the cell survival rate was 44%. Therefore, it can be concluded that co-culturing HUVECs with 0.6mM H2O2 for 1 h induced HUVEC cell damage, and a model of endothelial dysfunction was successfully established.

[0199] 3.2 Cell viability

[0200] Collect HUVEC cells at a ratio of 2 × 10⁻⁶. 4 Cells were incubated at a density of [number] cells / mL for 12-16 hours. When the cells reached 70-75% confluence, samples from Example 1 and Comparative Examples 3, 4, 8, and 9 (selected by CCK-8 assay) containing a safe concentration of hemoglobin-specific active peptides were added. A blank control group was also included. Each group had three replicates. Cells were incubated for 24 hours. After 24 hours of incubation, 0.6 mM H₂O₂ was added to each culture medium, and cell viability was assessed after another 1 hour of incubation.

[0201] Depend on Figure 1 It can be seen that the cell viability after intervention with the blood protein precise active peptide was higher than that of the Model, indicating that the blood protein precise active peptide has a protective effect against the reduction in cell viability caused by H2O2. Among them, the protective effects of Example 1, Comparative Example 3 and Comparative Example 4 are stronger, and the cell viability under the protective effect of these three blood protein precise active peptides can reach about 80%.

[0202] 3.3 Effects of blood protein-specific active peptides on mRNA expression of relevant target sites in an endothelial dysfunction model

[0203] Collect HUVEC cells at 2 × 10⁻⁶ 4Cells were seeded at a density of 10 cells / mL in 24-well plates and incubated for 24 h. When the cells reached 70%–75% confluence, samples from Example 1, Comparative Examples 3, 4, 8, and 9 (selected by CCK-8 screening) were added, along with a blank control group (control group) and an H2O2 group (model group). Each group had three replicates. After 24 h of incubation, 0.6 mM H2O2 was added and the cells were co-incubated for 1 h. Total RNA was then extracted from the cells using the Simply P Total RNA Extraction Kit (BioFlux). 2 µL of sterile, enzyme-free water was dropped onto a Nanodrop one plate to determine the RNA concentration and purity. A 20 µL qRT-PCR reaction system was established. The PCR amplification conditions were: pre-denaturation 95°C for 30 s; denaturation 95°C for 15 s; annealing 54°C for 30 s; extension 72°C for 45 s; for 40 cycles. The melting curve reaction conditions were: 95℃ for 30 s, 60℃ for 1 min, with fluorescence signals collected every 5℃ increase. Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., using GAPDH as an internal control. Primer sequence information is as follows:

[0204] GAPDH, 5′-CTATAAATTGAGCCCGCAGC-3′ (F) and 5′-GACCAAATCCGTTGACTCCG-3′ (R);

[0205] PI3K, 5′-GATTTTACCATGAGACCCAGAAC-3′ (F) and 5′-GCTTTTTCGTAAATCATCTGTGA-3′ (R);

[0206] AKT1, 5′-CCTGTGTCCGACCTGGACTAC-3′ (F) and 5′-CGGGTCAGGTACGGGCGTAT-3′ (R);

[0207] eNOS, 5′-TCCCTGCTGGCTTTGCTC-3′ (F) and 5′-AGGGGGCAGGCTGTGTTG-3′ (R).

[0208] It should be noted that all of the above primers are existing primers.

[0209] Figure 2 The effect of hemoglobin-specific active peptides on the expression level of eNOS mRNA in HUVEC cells. Figure 3 The effect of hemoglobin-specific active peptides on the expression level of AKT1 mRNA in HUVEC cells. Figure 4 The effect of hemoglobin-specific active peptides on the expression level of PI3K mRNA in HUVEC cells.

[0210] The three genes eNOS (endothelial nitric oxide synthase), AKT1 (protein kinase B), and PI3K (phosphatidylinositol 3-kinase) play key roles in vascular biology, cell signaling, and metabolic regulation. They are mainly involved in the PI3K / AKT / eNOS signaling pathway, affecting vasodilation, angiogenesis, endothelial cell survival, and inflammation regulation.

[0211] eNOS catalyzes the production of the vasodilating molecule NO (nitric oxide) from L-arginine, maintaining vascular endothelial function, inhibiting platelet aggregation and inflammatory responses, and promoting angiogenesis. AKT1 is a core molecule in the PI3K / AKT / mTOR (mammalian target of rapamycin) signaling pathway, regulating cell survival, proliferation, and metabolism. It promotes vasodilation by phosphorylating eNOS (activating NO production). PI3K catalyzes the conversion of PIP2 (phosphatidylinositol-4,5-bisphosphate) to PIP3 (phosphatidylinositol-3,4,5-triphosphate), activating AKT signaling. All these processes regulate cell growth, metabolism, survival, and angiogenesis.

[0212] The relationship among the three is as follows: PI3K is activated by growth factors to generate PIP3; AKT1 is recruited to the membrane by PIP3 and activated by phosphorylation of PDK1 / mTORC2; AKT1 phosphorylates eNOS, increasing NO production, which can cause vasodilation. NO promotes vasodilation, inhibits platelet aggregation, and has anti-inflammatory effects.

[0213] Compared to the Model group, the expression level of the eNOS gene in Example 1 was significantly increased, indicating that the intervention with the blood protein-specific active peptide in Example 1 could partially restore pathway activity, hence the higher eNOS expression compared to the Model group. Compared to the Model group and the control group, the expression level of the AKT1 gene in Example 1 and Comparative Examples 3, 4, 8, and 9 was significantly increased and basically the same as that in the control group, indicating that the intervention specifically activated the PI3K / AKT pathway. This is because AKT1, as a key signaling node, may rapidly recover its expression or phosphorylation after intervention with the blood protein-specific active peptide. Compared to the Model group and the control group, the expression of the PI3K gene in Example 1 and Comparative Example 3 was significantly increased, indicating that the blood protein-specific active peptide in Example 1 and Comparative Example 3 effectively cleared the inhibition of PI3K gene expression by H2O2.

[0214] The combined expression data of the three genes shows that the expression of PI3K and AKT1 genes after intervention with the precise active peptide of hemoglobin in Example 1 can basically recover to the level of the blank group, indicating that the PI3K-AKT axis function is intact. However, the recovery of eNOS has a certain lag, which may be because eNOS is also regulated by other factors.

[0215] 3.4 Effects of blood protein-specific active peptides on NOS3 and ET-1 levels in an endothelial dysfunction model

[0216] Collect HUVEC cells at 2 × 10⁻⁶ 4 Cells were seeded at a density of [number] cells / mL in 24-well plates and incubated for 24 h. When the cells reached 70%–75% confluence, samples from Example 1, Comparative Examples 3, 4, 8, and 9 (selected using CCK-8 assays) and a blank control group (control group) and an H2O2 group (model group) were added. Each group had three replicates. Cells were incubated for 24 h, followed by co-incubation with 0.6 mM H2O2 for 1 h. The cell supernatant was collected, centrifuged at 10,000 rpm / min for 20 minutes at 4°C to remove cell debris, and stored at -80°C for later use. Measurements were performed according to the kit (Elabscience Biotechnology Co., Ltd.).

[0217] Figure 5 To investigate the effect of the blood protein-specific active peptide on NOS3 levels in an endothelial dysfunction model, the NOS3 levels in Example 1 and Comparative Examples 3, 4, 8, and 9 were all higher than the model group but lower than the control group. This indicates that the blood protein-specific active peptide partially restored the inhibitory effect of H2O2 on NOS3, but did not completely restore it to normal levels. This is consistent with the results of eNOS gene expression. Among them, Comparative Example 9 had the highest NOS3 level, indicating that the blood protein-specific active peptide in Comparative Example 9 restored the inhibitory effect of H2O2 on NOS3. O It has the strongest inhibitory effect on NOS3;

[0218] ET-1 is a potent vasoconstrictive peptide secreted by endothelial cells, involved in the regulation of vascular tone, cell proliferation, and inflammatory responses. Figure 6 To investigate the effect of blood protein-specific active peptides on ET-1 levels in an endothelial dysfunction model, by... Figure 6 It can be seen that the ET-1 content in Example 1 is lower than that in the model group and relatively equal to that in the blank group, indicating that the blood protein precise active peptide in Example 1 can completely reverse the H2O2-induced increase in ET-1. In contrast, the ET-1 content in Comparative Examples 3, 4, 8, and 9 is lower than that in the model group and higher than that in the blank group, indicating that the blood protein precise active peptide in Comparative Examples 3, 4, 8, and 9 can partially reverse the H2O2-induced increase in ET-1.

[0219] In summary, the expression of eNOS, AKT1, and PI3K genes in HUVEC cells induced by H2O2 after intervention in Example 1 was increased, NOS3 content increased, and ET-1 content decreased. This indicates that the blood protein-specific active peptide in Example 1 improves endothelial dysfunction caused by oxidative stress, improves vasodilation, inhibits vasoconstriction and remodeling, and protects the vascular endothelial barrier by activating the PI3K / AKT1 / eNOS signaling pathway.

[0220] 4. Experiment on the Qi- and Blood-Nourishing Efficacy of Blood Protein Precision Active Peptides

[0221] 4.1 Testing Materials

[0222] 4.1.1 Sample Preparation Information

[0223] Example 1: Precisely Active Peptides for Blood Proteins (hereinafter referred to as blood protein precise active peptide), the solvent is standard dilution water.

[0224] 4.1.2 Laboratory Animals

[0225] Zebrafish were raised in aquarium water at 28℃ (water quality: 200 mg of instant sea salt added per 1 L of reverse osmosis water, conductivity 450~550 μS / cm; pH 6.5~8.5; hardness 50~100 mg / L CaCO3), bred and provided by our aquarium. The laboratory animal use license number is: SYXK (Zhejiang) 2022-0004. The husbandry and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2025-10695-01.

[0226] 4.1.3 Instruments, Consumables and Reagents

[0227] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China).

[0228] Phenylated hydrazine (batch number YH0170509, Shanghai Yihe Biotechnology Co., Ltd., China); o-anisidine (batch number MKBX3619V, Sigma, USA); anhydrous sodium acetate (batch number F20090306, Sinopharm Chemical Reagent Co., Ltd., China); anhydrous ethanol (batch number 20220414, Shanghai Aladdin Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland).

[0229] 4.2 Detection Methods

[0230] 4.2.1 MTC Measurement

[0231] Wild-type AB strain zebrafish with a 3-day pf (dpf) count were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The samples were administered in water (concentrations shown in Table 4). A normal control group and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for 30 h, except for the normal control group, all other experimental groups were administered phenylhydrazine in water to establish a zebrafish anemia model. Treatment continued at 28℃ for another 18 h, and zebrafish mortality was observed and recorded to determine the mean toxicity (MTC) of the samples in the model zebrafish.

[0232] 4.2.2. Evaluation of Qi and Blood Tonifying Effects

[0233] Wild-type AB strain zebrafish (3 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. The samples were administered in water (concentrations shown in Table 5). A normal control group and a model control group were also included, with a volume of 3 mL per well. After treatment at 28℃ for 30 h, except for the normal control group, all other experimental groups were given phenylhydrazine in water to establish a zebrafish anemia model. After another 18 h of treatment at 28℃, o-anisidine staining was performed. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were collected using advanced image processing software, and the staining intensity of erythrocytes in the zebrafish heart was analyzed. The statistical significance of this index was used to evaluate the blood-tonifying efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using software, and p-values ​​were calculated. A value of 0.05 indicates that the difference is statistically significant.

[0234] 4.3 Test Results

[0235] 4.3.1 MTC

[0236] Under the conditions of this experiment, the MTC of the blood protein-specific active peptide for replenishing qi and blood was 250 μg / mL. See Table 4 for details.

[0237] Table 4 Results of the experiment on the concentration of samples for replenishing qi and blood (n = 30)

[0238]

[0239] 4.3.2 Evaluation of Qi and Blood Tonifying Efficacy

[0240] Under the experimental conditions, the blood protein-specific active peptides exhibited the effect of replenishing qi and blood. See Table 5 for details. Figure 7 and Figure 8 .

[0241] Table 5. Experimental results evaluating the qi-tonifying and blood-nourishing effects of the samples (n = 10)

[0242]

[0243] Figure 7 This is a typical image of the staining intensity of red blood cells in zebrafish heart after sample processing (Note: The yellow dashed box represents the zebrafish heart as the analyzed site). Figure 8 The staining intensity of zebrafish heart erythrocytes after sample treatment was compared with that of the model control group. p 0.001.

[0244] 5. Experimental study on the efficacy of blood protein-specific active peptides in improving microcirculation.

[0245] 5.1 Testing Materials

[0246] 5.1.1 Sample Preparation Information

[0247] Blood protein precise active peptides, with standard dilution water as the solvent.

[0248] 5.1.2 Laboratory Animals

[0249] Zebrafish were raised in aquarium water at 28℃ (water quality: 200 mg of instant sea salt added per 1 L of reverse osmosis water, conductivity 450~550 μS / cm; pH 6.5~8.5; hardness 50~100 mg / L CaCO3), bred and provided by our aquarium. The laboratory animal use license number is: SYXK (Zhejiang) 2022-0004. The husbandry and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2025-10695-01.

[0250] 5.1.3 Instruments, Consumables and Reagents

[0251] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Beilanbo Biotechnology Co., Ltd., China); Cardiac blood flow analyzer (ZebraBlood 3.4, ViewPoint Life Sciences, France).

[0252] Methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); Ponatinib (batch number F1511088, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0253] 5.2 Detection Methods

[0254] 5.2.1 Determination of Maximum Detectable Concentration (MTC)

[0255] Wild-type AB strain zebrafish, 5 days post-fertilization (5 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water (concentrations shown in Table 6), and a normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were administered ponatinib in water to establish a zebrafish endothelial vascular injury model. After treatment at 28℃ for 1 day, the MTC of the samples in the model zebrafish was measured.

[0256] 5.2.2 Improves microcirculation

[0257] Wild-type AB strain zebrafish with a 5 dpf growth rate were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered in water (concentrations shown in Table 7), and a normal control group and a model control group were also included. The volume of each well was 3 mL. Except for the normal control group, all other experimental groups were administered ponatinib in water to establish a zebrafish vascular endothelial injury model. After treatment at 28℃ for 1 day, 10 zebrafish from each experimental group were randomly selected and placed in a cardiac blood flow analysis system to detect the blood flow velocity. The statistical analysis results of this index were used to evaluate the efficacy of the sample in improving microcirculation. Statistical results are expressed as mean ± SE. Statistical analysis was performed using software, and p-values ​​were used. A value of 0.05 indicates that the difference is statistically significant.

[0258] 5.3 Test Results

[0259] 5.3.1 MTC

[0260] Under the conditions of this experiment, the MTC of the blood protein-specific active peptide in improving microcirculation was 62.5 μg / mL. See Table 6 for details.

[0261] Table 6 Results of the experiment on the concentration of the sample that improves microcirculation (n = 30)

[0262]

[0263] 5.3.2 Improves microcirculation

[0264] Under the experimental conditions, the precise bioactive peptides of hemoproteins improved microcirculation, specifically by increasing blood flow velocity. See Table 7 for details. Figure 9 ,in Figure 9 The blood flow velocity of zebrafish after sample treatment was compared with that of the model control group. p 0.05, p 0.001.

[0265] Table 7. Experimental results evaluating the efficacy of the samples in improving microcirculation (n = 10)

[0266]

[0267] 6. Peptide screening for precise bioactive peptides of blood proteins

[0268] 6.1 Pretreatment Method

[0269] 6.1.1 Sample Dissolution

[0270] 1) Weigh 5.4 mg of sample 3, add 540 μL of water, and shake to mix.

[0271] 6.1.2 Reductive Alkylation

[0272] 1) Take 20 μL of sample, add water to make up to 100 μL, and set aside;

[0273] 2) Accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L, and reduce it in a 56℃ water bath for 1 h;

[0274] 3) Accurately pipette 2 μL of 1M IAM solution into the sample to make the final IAM concentration 20 mmol / L, and react in the dark at room temperature for 40 min.

[0275] 4) Accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L, in order to neutralize unreacted IAM.

[0276] 6.1.3 Desalination

[0277] 1) Activation: Activate the desalting column twice with 300 μL of 100% ACN;

[0278] 2) Equilibration: Equilibrate the desalting column twice with 200 μL of 0.1% TFA;

[0279] 3) Sample loading: Load the sample twice;

[0280] 4) Desalting: Desalting three times with 200 μL of 0.1% TFA;

[0281] 5) Elution: Elute once with 300 μL 80%ACN-20% 0.1%TFA, and dry under vacuum at 45℃.

[0282] 6.2 Computer Usage Requirements

[0283] 6.2.1 Liquid Chromatography Conditions

[0284] 1) Pre-column: 150 μm id × 50 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm; Analytical column: 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm

[0285] 2) Mobile phase A: 0.1% FA;

[0286] 3) Mobile phase B: 0.1% FA, 80% ACN;

[0287] 4) Flow rate: 600 nL / min;

[0288] 5) Analysis time for each component: 66 min;

[0289] 6) Specific chromatographic conditions are shown in Table 8:

[0290] Table 8 Chromatographic conditions

[0291]

[0292] 6.2.2 Mass Spectrometry Conditions

[0293] Level 1 mass spectrometry parameters:

[0294] 1) Resolution: 70,000

[0295] 2) AGCtarget: 3e6

[0296] 3) Maximum IT: 100ms

[0297] 4) Scanrange: 100 to 1500 m / z

[0298] Secondary mass spectrometry parameters:

[0299] 1) Resolution: 17,500

[0300] 2) AGCtarget: 1e5

[0301] 3) Maximum IT: 50ms

[0302] 4) TopN: 20

[0303] 5) NCE / Stepped NCE: 28

[0304] Raw data was obtained through mass spectrometry.

[0305] 6.3 Search Criteria

[0306] The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows:

[0307] 1) Fixed modifications: Carbamidomethyl (C).

[0308] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term).

[0309] 3) Enzyme: Non specific.

[0310] 4) Database: uniprotkb_taxonomy_id_9823_AND_reviewed_2025_03_14.fasta.

[0311] 5) Peptide Mass Tolerance: 20 ppm

[0312] 6) Secondary mass spectrometry bias (Fragment Mass Tolerance): 0.02 Da

[0313] Raw files acquired by mass spectrometry were retrieved from a database, and peptides with an abundance greater than 10,400,000 were selected. Activity prediction was then performed on these fragments, and the results were chosen. Peptides with a concentration of 0.7 g / L were screened using abundance ranking, resulting in 13 peptides. A peptide database search revealed that 12 of these peptides were novel and not previously independently reported (except for peptide VVYP, which was publicly known). Toxicity prediction showed that all 12 were non-toxic. Related peptide information is listed in Table 9.

[0314] Table 9 Peptide Information

[0315]

[0316] Note: Peptide ProteinMetrics Confidential : Amino acid sequence of the identified peptide; Mass: molecular weight of the peptide; Score: activity prediction score; Lenth: number of amino acids in the peptide; RT: retention time of the peptide, in min; -10 lgP: significance score of the peptide identified by database search or de novo sequencing; Intensity: peptide abundance.

[0317] Liquid chromatography-mass spectrometry (LC-MS) results showed that the contents of active tetrapeptides such as LGFP and VVYP were 3.69% and 0.88%, respectively (the proportion of the target peptide to the total peptide content). Since the antihypertensive activity of VVYP has been confirmed by multiple studies, its identity as an antihypertensive peptide is already clear, and therefore it was not included in subsequent validation work.

[0318] 7. Molecular docking

[0319] eNOS (PDB ID: 3NOS), AKT1 (PDB ID: 3O96), PI3K (PDB ID: 4L23), and ETA receptor (PDB ID: 5GLH) are four important targets, each playing a key role in the treatment of cardiovascular disease, cancer, and hypertension.

[0320] eNOS (endothelial nitric oxide synthase) regulates vasodilation by catalyzing the production of nitric oxide (NO) from L-arginine. On the one hand, it can lower blood pressure, and on the other hand, vasodilation can increase blood flow, thereby improving microcirculation.

[0321] AKT1 (protein kinase B) is a "super helper" of eNOS, which can enhance the efficiency of eNOS and enable more NO to be produced.

[0322] PI3K (phosphatidylinositol 3-kinase) mainly functions through the PI3K / AKT / eNOS pathway. PI3K transmits a signal (PIP3) to AKT1, and AKT1 activates eNOS (phosphorylation) upon receiving the signal, thereby increasing NO production.

[0323] ETA receptors (endothelin receptor A) act as "constriction switches" in blood vessels, narrowing them and raising blood pressure. When endothelin-1 (ET-1) (a powerful vasoconstrictor) binds to ETA receptors, it causes the vascular muscles to tighten, narrowing the blood vessels. At the same time, it also inhibits eNOS, reducing NO release and further aggravating vasoconstriction.

[0324] G-CSF (Granulocyte Colony-Stimulating Factor) (PDB:1CD9) is a key hematopoietic growth factor that primarily promotes the proliferation, differentiation, and survival of neutrophils.

[0325] GATA1 (GATA Binding Protein 1, a zinc finger transcription factor) is a key transcription factor in the development of the hematopoietic system. Its main functions include: erythropoiesis (regulating the expression of hemoglobin synthesis genes such as α- and β-globin); megakaryocyte differentiation (affecting platelet production); and stem cell maintenance (participating in the self-renewal and directed differentiation of hematopoietic stem cells). In addition, GATA1 improves the state of qi and blood deficiency by activating erythrocyte-specific genes (such as EPO receptor and heme synthase).

[0326] Molecular docking software was used to dock the 12 selected fragments with the above six targets. The binding energies of peptides to receptors are summarized in Tables 10 to 12. The three-dimensional structures of eNOS (PDB: 3NOS), AKT1 (PDB: 3O96), PI3K (PDB: 4L23), ETA receptor (PDB: 5GLH), G-CSF (PDB: 1CD9), and GATA1 (PDB: 2FIV) were downloaded from the database.

[0327] Table 10. Binding energies of peptides to eNOS and AKT1 receptors

[0328]

[0329] Table 11 Binding energies of peptides to PI3K and ETA receptors

[0330]

[0331] Table 12 Binding energies of peptides to G-CSF and GATA1 receptors

[0332]

[0333] eNOS regulates vasodilation by catalyzing the production of nitric oxide (NO) from L-arginine. As shown in Table 10, all 12 peptides have a certain binding energy with eNOS. After the peptides bind to the receptor, they activate eNOS, promote NO production, and promote vasodilation. This has potential therapeutic significance for hypertension or cardiovascular diseases. At the same time, vasodilation can also increase blood flow velocity, thereby improving microcirculation.

[0334] Peptides 1-2 and 4-11 can bind to the protein receptors of AKT1 and PI3K, indicating that the PI3K / AKT / eNOS pathway is activated. The peptides bind to the AKT1 receptor, enhancing its activity by phosphorylating eNOS (Ser1177) and promoting NO production. The peptides bind to the PI3K receptor, further supporting the activation of the PI3K / AKT / eNOS pathway. The mechanism may involve helping PI3K anchor to the cell membrane, increasing PIP3 production efficiency, or enhancing PI3K catalytic activity. PI3K generates PIP3, recruiting and activating AKT1 to the cell membrane, thus enhancing pathway efficiency.

[0335] Peptides 1-2 and 4-12 can both bind to the ETA receptor, which belongs to the GPCR family and mediates the vasoconstrictive effect of ET-1, closely related to pulmonary hypertension. The binding of these peptides to the ETA receptor may block the ET-1 / ETA signaling, inhibiting ET-1 transcription or secretion, leading to a decrease in intracellular ET-1 levels. Reduced ET-1 levels decrease vascular smooth muscle contraction and improve endothelium-dependent vasodilation.

[0336] The above results indicate that peptides 1-2 and 4-11 improve endothelial function and microcirculation through dual regulation (pro-relaxation + anti-contraction).

[0337] Peptides 1-12 can all bind to G-CSF receptors, mimicking or enhancing the physiological functions of G-CSF, thereby promoting hematopoiesis (especially the proliferation and differentiation of neutrophil lineage).

[0338] Peptides 1-12 can all bind to GATA1, suggesting that these peptides may precisely regulate GATA1 activity, thereby promoting the production of red blood cells and hemoglobin and improving anemia. By enhancing the binding efficiency of GATA1 to target genes or protecting it from degradation, the efficiency of hematopoietic stem cell differentiation into mature red blood cells can be improved, while optimizing the expression of iron metabolism-related genes, thus addressing the problem of "insufficient blood and qi" at the molecular level.

[0339] Further screening identified five sequences—LGFP, IGFP, LFL, LFI, and FESF—as characteristic peptide sequences among the blood protein's precise active peptides, which have the effects of replenishing qi and blood, improving microcirculation, and promoting vasodilation.

[0340] Figure 10 This is a 2D diagram showing the docking of LGFP with eNOS molecules. Figure 11 This is a 2D diagram showing the docking of IGFP with the eNOS molecule. Figure 12 A 2D diagram showing the docking of LFL with eNOS molecules; Figure 13 A 2D diagram showing the docking of LFI with eNOS molecules; Figure 14 A 2D diagram showing the docking of FESF and eNOS molecules; Figure 15This is a 2D diagram of the docking of LGFP with AKT1 molecules; Figure 16 This is a 2D diagram of the docking of IGFP with AKT1 molecules; Figure 17 A 2D diagram showing the docking of LFL and AKT1 molecules; Figure 18 A 2D diagram showing the docking of LFI and AKT1 molecules; Figure 19 A 2D diagram showing the docking of FESF with AKT1 molecules; Figure 20 This is a 2D diagram of the docking of LGFP with PI3K molecules; Figure 21 This is a 2D diagram of the docking of IGFP and PI3K molecules. Figure 22 A 2D diagram showing the docking of LFL and PI3K molecules; Figure 23 A 2D diagram showing the docking of LFI and PI3K molecules; Figure 24 A 2D diagram showing the docking of FESF with PI3K molecules; Figure 25 This is a 2D diagram of the docking of LGFP with the ETA receptor molecule. Figure 26 This is a 2D diagram of the docking of IGFP with the ETA receptor molecule. Figure 27 A 2D diagram showing the docking of LFL with the ETA receptor molecule; Figure 28 A 2D diagram showing the docking of LFI with the ETA receptor molecule; Figure 29 This is a 2D diagram of the docking of FESF with the ETA receptor molecule. Figure 30 This is a 2D diagram showing the docking of LGFP with the G-CSF receptor molecule. Figure 31 This is a 2D diagram showing the docking of IGFP with the G-CSF receptor molecule. Figure 32 A 2D diagram showing the docking of LFL with the G-CSF receptor molecule; Figure 33 A 2D diagram showing the docking of LFI with the G-CSF receptor molecule; Figure 34 This is a 2D diagram of the docking of FESF with the G-CSF receptor molecule. Figure 35 This is a 2D diagram of the docking of LGFP with the GATA1 receptor molecule. Figure 36 This is a 2D diagram of the docking of IGFP with the GATA1 receptor molecule. Figure 37 This is a 2D diagram of the docking of LFL with the GATA1 receptor molecule. Figure 38 This is a 2D diagram of the docking of LFI with the GATA1 receptor molecule. Figure 39 This is a 2D diagram of the docking of FESF with the GATA1 receptor molecule.

[0341] like Figure 10 As shown, LGFP and eNOS interact through carbon-hydrogen bonds, van der Waals forces, salt bridges, attractive forces, and covalent hydrogen bonds; as Figure 11 As shown, IGFP and eNOS interact through carbon-hydrogen bonds, van der Waals forces, salt bridges, attractive forces, and covalent hydrogen bonds; as Figure 12 As shown, LFL and eNOS interact through carbon-hydrogen bonds, van der Waals forces, and salt bridges; as Figure 13 As shown, LFI and eNOS interact through covalent hydrogen bonds, van der Waals forces, and salt bridges; as Figure 14 As shown, FESF and eNOS interact through carbon-hydrogen bonds, van der Waals forces, salt bridges, attractive forces, and covalent hydrogen bonds.

[0342] like Figure 15 As shown, LGFP and AKT1 interact through carbon-hydrogen bonds, van der Waals forces, salt bridges, and covalent hydrogen bonds; as Figure 16 As shown, IGFP and AKT1 interact through carbon-hydrogen bonds, van der Waals forces, attractive forces, and covalent hydrogen bonds; as Figure 17 As shown, LFL and AKT1 interact with each other through attractive and van der Waals forces; as Figure 18 As shown, LFI and AKT1 interact through carbon-hydrogen bonds, attractive forces, van der Waals forces, and salt bridges; as Figure 19 As shown, FESF and AKT1 interact through carbon-hydrogen bonds, van der Waals forces, attractive forces, and covalent hydrogen bonds.

[0343] like Figure 20 As shown, LGFP and PI3K interact via carbon-hydrogen bonds and van der Waals forces; Figure 21 As shown, IGFP and PI3K interact through carbon-hydrogen bonds, van der Waals forces, attractive forces, and covalent hydrogen bonds; as Figure 22 As shown, LFL and PI3K interact via covalent hydrogen bonds and van der Waals forces; Figure 23 As shown, LFI and PI3K interact through covalent hydrogen bonds and van der Waals forces; Figure 24 As shown, FESF and PI3K interact through carbon-hydrogen bonds, van der Waals forces, salt bridges, and covalent hydrogen bonds.

[0344] like Figure 25 As shown, LGFP interacts with the ETA receptor via C-H bonds, van der Waals forces, salt bridges, and covalent hydrogen bonds; Figure 26 As shown, IGFP interacts with the ETA receptor through van der Waals forces, attractive forces, and covalent hydrogen bonds; as Figure 27 As shown, LFL interacts with the ETA receptor through van der Waals forces, attractive forces, and covalent hydrogen bonds; Figure 28 As shown, LFI interacts with the ETA receptor via covalent hydrogen bonds and van der Waals forces; Figure 29 As shown, FESF interacts with the ETA receptor via carbon-hydrogen bonds, van der Waals forces, salt bridges, and covalent hydrogen bonds.

[0345] like Figure 30 As shown, LGFP interacts with the G-CSF receptor via carbon-hydrogen bonds, van der Waals forces, and covalent hydrogen bonds; Figure 31As shown, IGFP interacts with the G-CSF receptor via carbon-hydrogen bonds, van der Waals forces, and covalent hydrogen bonds; Figure 32 As shown, LFL interacts with G-CSF receptors through van der Waals forces, salt bridges, C-H bonds, and covalent hydrogen bonds; Figure 33 As shown, LFI interacts with the G-CSF receptor through covalent hydrogen bonds, van der Waals forces, and C-H bonds; Figure 34 As shown, FESF interacts with the G-CSF receptor via carbon-hydrogen bonds, van der Waals forces, and covalent hydrogen bonds.

[0346] like Figure 35 As shown, LGFP interacts with the GATA1 receptor via carbon-hydrogen bonds, attractive forces, van der Waals forces, and covalent hydrogen bonds; Figure 36 As shown, IGFP interacts with the GATA1 receptor through van der Waals forces, attractive forces, C-H bonds, and covalent hydrogen bonds; Figure 37 As shown, LFL interacts with the GATA1 receptor through van der Waals forces, attractive forces, C-H bonds, and covalent hydrogen bonds; Figure 38 As shown, LFI interacts with the GATA1 receptor through covalent hydrogen bonds, van der Waals forces, and C-H bonds; Figure 39 As shown, FESF interacts with the GATA1 receptor via carbon-hydrogen bonds, van der Waals forces, salt bridges, and covalent hydrogen bonds.

[0347] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing a blood protein-specific active peptide with functions of replenishing qi and blood, improving microcirculation, and promoting vasodilation, characterized in that, Includes the following steps: S1. Mix hemoglobin powder with water evenly and homogenize to obtain a pretreatment solution; S2. Add Bacillus subtilis neutral protease to the pretreatment solution for the first hydrolysis and enzyme inactivation to obtain the first hydrolysate. S3. Add acidic protease to the first hydrolysate for a second hydrolysis and enzyme inactivation to obtain a second hydrolysate; S4. The second hydrolysate is filtered, ultrafiltered and spray-dried to obtain blood protein precise active peptides; The mass ratio of hemoglobin powder to water in S1 is 1:(5~15); the homogenization treatment time is 30~90 min. The amount of neutral protease from Bacillus subtilis added is 3% to 4% of the mass of the hemoglobin powder; the amount of acidic protease added is 0.2% to 0.8% of the mass of the hemoglobin powder. The neutral protease of Bacillus subtilis has an enzyme activity of 80,000 U / g, and the acidic protease has an enzyme activity of 600,000 U / g. The temperature of the first hydrolysis is 50℃~55℃, the pH of the hydrolysis reaction is 6.5~7.0, and the hydrolysis time is 2~4h; the temperature of the second hydrolysis is 35℃~40℃, the pH of the hydrolysis reaction is 2.0~3.0, and the hydrolysis time is 2~4h. The enzyme inactivation conditions described in S2 and S3 are both at 85℃~90℃ for 15~20min. The filtration method described in S4 is solid-liquid separation. The ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 5000 Da.

2. The preparation method according to claim 1, characterized in that, The hemoprotein-specific active peptide contains at least one of LGFP, IGFP, FPHFN, LFL, LFI, FESF, ILF, LIF, FLL, FIL, FII, or FPHFD; The specific sequences of LGFP are shown in SEQ ID NO.1; IGFP is shown in SEQ ID NO.2; FPHFN is shown in SEQ ID NO.3; FESF is shown in SEQ ID NO.4; and FPHFD is shown in SEQ ID NO.

5.

3. The application of a blood protein-specific active peptide prepared by the method described in claim 1 in the preparation of functional products for replenishing qi and blood, improving microcirculation, or promoting vasodilation.

Images