Natto oligopeptide, manufacturing method thereof, and application thereof in thrombolysis and blood pressure reduction

Abstract

To provide Natto oligopeptide, to provide a manufacturing method thereof, and to provide an application thereof.SOLUTION: A manufacturing method of Natto antithrombotic oligopeptide includes determining characteristics of Natto antithrombotic peptides by searching a bioactive peptide database to obtain Natto-derived polypeptides with antithrombotic activity. Characteristic information includes the fact that a molecular weight is less than 2,000 Da, a type of an N-terminal amino acid residue is glycine G, a type of a C-terminal amino acid residue is lysine K or arginine R, and a net charge is ±2. Statistics are collected for the type and quantity of amino acid residues located in a front position of glycine G, and the type and quantity of amino acid residues located in a front position of arginine R for each peptide in a peptide solution. The peptides are ranked based on the statistical quantity, and the peptide bonds within the ranking are used as enzymatic cleavage sites, which are then cleaved using a complex protease. A low molecular weight peptide solution and a peptide solution with a net charge of ±2 are collected.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to the field of producing active peptides, and in particular to a method for producing natto oligopeptides and their application in thrombolysis and blood pressure reduction. [Background technology]

[0002] Thrombotic diseases, a typical cardiovascular disease, pose a serious threat to human life and health. They are characterized by high incidence, disability, and mortality rates, ranking among the highest among all diseases. They not only cause immense suffering to patients but also have a significant impact on their families. At the same time, a range of cardiovascular diseases, including thrombosis, are likely to be caused by persistent hypertension. Currently, various thrombolytic agents for thrombolytic treatment are available in clinical settings, including streptokinase, urokinase, staphylokinase, single-chain urokinase, and tissue-type plasminogen activator (t-PA). While these agents have high thrombolytic specificity, they are also prone to allergic reactions and severe bleeding.

[0003] Extensive research has demonstrated that natto has medical and health benefits, including preventing osteoporosis, promoting blood coagulation, lowering blood pressure, and improving blood sugar levels. The excellent health benefits of natto are closely related to the changes in the basic components of soybeans during its production process. Under the action of natto bacillus, complex biochemical reactions occur between soybean proteins, fats, starches, and isoflavones, preserving the original active components while also producing new active substances such as nattokinase (NK), antioxidant peptides, and antimicrobial peptides. Related research has shown that nattokinase has certain antiplatelet aggregation, ex vivo thrombolysis, blood pressure reduction, and blood lipid reduction effects. Its thrombolytic mechanism is that nattokinase directly hydrolyzes fibrin and plasmin substrates, thereby dissolving blood clots. It converts endogenous kinase to urokinase, which in turn degrades plasminogen activator inhibitors and increases the levels of plasminogen activators, thereby dissolving the clot.

[0004] However, research on natto components other than nattokinase, especially active peptide components, is still lacking. Yahui Song discovered RBPs (reactive blood pressure markers) with high ACE inhibitory activity by fermenting natto. After characterizing these RBPs, he found that they significantly reduced the incidence of spontaneously hypertensive rats and had protective effects on the kidney, thoracic aorta, and heart. Kousuke Sato's research confirmed the in vitro DPPIV inhibitory activity of natto isolated peptides. These studies have revealed that natto contains a variety of active peptide components with physiological activities. In Chinese patent application CN202310653994.6, entitled "Method and Product for Producing Natto and Natto Active Peptides," soybeans were fermented by inoculation with a complex fungus. Mature fresh natto was ground into a semi-liquid state, then dried and re-ground to obtain natto active peptides. While the above studies only isolated active peptide components specific to natto products, there has been no further exploration or utilization of natto protein / active peptide resources. Chinese patent application CN202311164212.9, entitled "Method for producing natto lipid-lowering peptides and their applications," and Chinese patent application CN202311164254.2, entitled "Method for producing natto antioxidant peptides and beverages using the same," disclose a method for obtaining specific active natto peptides through enzymatic hydrolysis, but do not address natto's most notable antithrombotic effect. Furthermore, the acid-base environment during enzymatic hydrolysis and the high temperature environment during enzyme removal are highly likely to destroy the activity of nattokinase. Lee KA et al. isolated two antithrombotic peptide segments, SSGE and DEE, from soy protein and demonstrated their inhibitory effect on platelet aggregation. Although some changes in the soy protein components occur during natto fermentation, there is reason to believe that natto has the potential to produce antithrombotic peptides. Currently, the most common natto product on the market other than natto is freeze-dried natto powder, which is divided into different specifications based on nattokinase activity. Research has been intensively conducted on ways to further increase the nattokinase content by improving the fermentation process, but the development and use of ingredients other than nattokinase is very limited.How to develop active ingredients other than nattokinase to enhance the improving effects of natto products on thrombotic cardiovascular diseases is extremely important for maximizing the utilization of natto resources. Summary of the Invention

[0005] In order to solve the problem of obtaining functional oligopeptides and further the problem of obtaining polypeptides derived from natto having antithrombotic activity, in a first aspect, according to the method for producing oligopeptides of some embodiments of the present application, Searching a biologically active peptide database to determine peptide characteristics related to the functionality of a biological target, the characteristics including molecular weight distribution, type of N-terminal amino acid residue, type of C-terminal amino acid residue, and net charge; A biological object is prepared in a peptide solution, and the type and quantity of amino acids preceding a specific N-terminal amino acid of each peptide in the peptide solution are separately calculated; cleaving the enzyme cleavage sites, which are all or a part of the peptide bond between a specific N-terminal amino acid and the amino acid preceding it and / or the carboxyl terminal peptide bond of a C-terminal amino acid, using a conjugated protease; filtering the solution of cleaved peptides to collect a solution of peptides having a molecular weight that matches the molecular weight distribution in said characteristic; Separating and purifying a solution of peptides having a molecular weight that matches the molecular weight distribution in the characteristics, and collecting a solution of peptides having a net charge that matches the characteristics, to obtain the oligopeptide; Includes.

[0006] In a second aspect, according to the method for producing an oligopeptide of some embodiments of the present application, Searching a biologically active peptide database to determine the characteristics of peptides related to the antithrombotic function of natto, the determined characteristic information including that the molecular weight is less than 2000 Da, the type of the N-terminal amino acid residue is glycine G, the type of the C-terminal amino acid residue is lysine K or arginine R, and the net charge is ±2; preparing a solution of natto peptides, and statistically calculating the type and number of amino acid residues located in front of glycine G and the type and number of amino acid residues located in front of arginine R of each peptide in the peptide solution; Ranking the samples based on the statistical quantity, and then, based on the ranking, using a composite protease to cleave the tyrosine-glycine peptide bond, the phenylalanine-glycine peptide bond, the tyrosine-arginine peptide bond, the lysine carboxyl terminal peptide bond, and the arginine carboxyl terminal peptide bond as enzymatic cleavage sites, the composite protease being composed of trypsin and chymotrypsin, the ratio of trypsin:chymotrypsin being 1:(1-3), the enzymatic decomposition temperature being 37°C to 45°C, the enzymatic decomposition pH being 7.5 to 8.5, and the enzymatic decomposition time being 3 hours to 6 hours; filtering the solution of cleaved peptides and collecting a solution of peptides having a molecular weight of less than 2000 Da; Separating and purifying a solution of peptides having a molecular weight of less than 2000 Da, and collecting a solution of peptides having a net charge of ±2 to obtain a first oligopeptide; Includes.

[0007] According to some embodiments of the present application, the method for producing an oligopeptide further includes mixing the solution of the peptide with a net charge of ±2 with a solution of a nattokinase component to obtain a second oligopeptide; Preparing the nattokinase component solution includes passing the natto powder solution through an ultrafiltration membrane with a molecular weight cut off (MWCO) of 29,000 Da to obtain a first permeate, and passing the first permeate through an ultrafiltration membrane with a molecular weight cut off of 26,000 Da to obtain a second permeate and a second intercept liquid, where the second intercept liquid is the nattokinase component solution.

[0008] According to the method for producing an oligopeptide of some embodiments of the present application, natto is produced from soybeans.

[0009] According to some embodiments of the method for producing an oligopeptide of the present application, the method further comprises freeze-drying the first oligopeptide to obtain natto oligopeptide powder.

[0010] According to some embodiments of the method for producing an oligopeptide of the present application, the method further comprises freeze-drying the second oligopeptide to obtain natto oligopeptide powder.

[0011] According to some embodiments of the method for producing oligopeptides of the present application, preparing a solution of small natto peptides includes adding digestive enzymes to the first permeate and the second blocking solution to obtain the solution of small natto peptides.

[0012] According to the method for producing an oligopeptide of some embodiments of the present application, the digestive enzyme is trypsin.

[0013] According to some embodiments of the method for producing an oligopeptide of the present application, filtering the solution of cleaved peptides includes filtering the solution of cleaved peptides through a nanofiltration membrane having a molecular weight cutoff of 2000 Da, and the permeate of the nanofiltration membrane is a solution of peptides having a molecular weight of less than 2000 Da; Separating and purifying a solution of peptides having a molecular weight of less than 2000 Da includes separating and purifying a solution of peptides having a molecular weight of less than 2000 Da by ion exchange chromatography.

[0014] According to the method for producing an oligopeptide of some embodiments of the present application, the ion exchange chromatography comprises a strong cation exchange column.

[0015] In some embodiments of the method for producing an oligopeptide according to the present application, the ion exchange chromatography includes an SP Sepharose High Performance strong cation exchange column. After loading the sample, the strong cation exchange column is equilibrated with 20 mmol / L PB (pH 6.0) and eluted with a 0-40 min: 0-1 M NaCl gradient. The eluent is collected for 11-19 min to obtain a solution of the peptide with a net charge of ±2.

[0016] According to some embodiments of the method for producing an oligopeptide of the present application, the peptides in a natto peptide solution are sequenced, and based on the sequencing, the type and number of amino acid residues located in front of glycine G and the type and number of amino acid residues located in front of arginine R of each peptide in the peptide solution are statistically determined.

[0017] Ranking based on statistical quantities is ranking different types of pre-glycine G amino acid residues by number; Ranking different types of amino acid residues preceding arginine R by number; based on the ranking of the number of amino acid residues preceding the different types of glycine G, cleaving the peptide bond between the type of amino acid residue with the largest number or the type with the first number ranking and glycine G to obtain a peptide whose N-terminal amino acid residue type is glycine G; cleaving the peptide bond between the type of amino acid residue with the largest number or the type with the first number ranking and arginine R based on the ranking of the number of amino acid residues preceding different types of arginine R to obtain a peptide whose N-terminal amino acid residue type is arginine R; cleaving the lysine carboxyl-terminal peptide bond to obtain a peptide in which the type of C-terminal amino acid residue is lysine K; cleaving the arginine carboxyl-terminal peptide bond of a peptide whose non-N-terminal amino acid residue is arginine R to obtain a C-terminal amino acid residue whose type is arginine R; and cleaving the arginine R of a peptide whose N-terminal amino acid residue is arginine R to obtain free L-arginine; Includes.

[0018] In a third aspect, an oligopeptide is produced by the method according to any one of the embodiments of the present application.

[0019] In a fourth aspect, there is provided a use of an oligopeptide according to some embodiments of the present application in the manufacture of a thrombolytic drug, or a use of an oligopeptide according to some embodiments of the present application in the manufacture of an antihypertensive drug, or a use of an oligopeptide according to some embodiments of the present application in the manufacture of a thrombolytic and antihypertensive drug.

[0020] Beneficial Effects: (1) In the first aspect of the present invention, the physicochemical properties of functional peptide segments are analyzed using an online database, and the molecular weight distribution, N-terminal amino acid residue type, C-terminal amino acid residue type, and net charge are selected as characteristics, and the enzymatic cleavage site is determined. For small peptides that meet the requirements of the characteristic molecular weight and net charge, the enzymatic cleavage site is cleaved with a complex protease, and then filtered, separated, and purified to obtain small peptides with the corresponding characteristics, allowing them to express the target functionality. (2) In an embodiment of the present invention, the physicochemical properties of antithrombotic peptide segments are analyzed using an online database, and peptide segments with antithrombotic activity are extracted from natto as much as possible. Nattokinase components are then added to further enhance the thrombolytic activity of the freeze-dried natto powder raw material. In this process, the amount of L-arginine (abbreviated as L-Arg) released in the raw material is increased, which further achieves blood pressure regulation through vasodilation, reduces vascular endothelial damage caused by high blood pressure and the increased risk of thrombosis due to the activation of the internal and external coagulation system, and alleviates the symptoms of thrombotic cardiovascular diseases such as atherosclerosis. (3) L-Arg is the only substrate for the production of nitric oxide (NO) in all mammals. NO can promote vasodilation, increase blood flow, improve blood circulation, regulate blood pressure, inhibit platelet aggregation and coagulation, and reduce the risk of thrombosis. Animal experiments and clinical studies have shown that L-Arg exerts certain effects on platelets, the coagulation process, and the fibrinolytic system through the NO pathway. The present invention uses multi-enzyme cleavage to increase the amount of specific small peptides and achieve the desired function. Furthermore, by taking into account the effect of L-Arg in cleavage, a multi-enzyme is designed to achieve targeted cleavage of specific small peptides while simultaneously increasing the amount of free L-Arg. This invention has both a thrombolytic effect and a hypotensive effect, which can reduce the risk of thrombosis. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1 shows the distribution of amino acid residues preceding glycine. [Figure 2] FIG. 2 shows the distribution of amino acid residues preceding arginine. [Figure 3] Figure 3 shows the distribution of N-terminal amino acid residue types. [Figure 4] FIG. 4 shows the distribution of C-terminal amino acid residue types. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be further illustrated by the following examples, which do not constitute limitations on the protection scope of the present invention.

[0023] The present invention provides a method for producing natto oligopeptide and its application in thrombolysis and blood pressure reduction. The method for producing natto oligopeptide includes the following steps:

[0024] S1: Freeze-dried natto powder produced by fermentation with Bacillus subtilis natto is used as raw material, dissolved in saline, and then separated using an ultrafiltration membrane to obtain a solution of components with molecular weights between 26,000 and 29,000 Da, which is called nattokinase component F1. After obtaining nattokinase component F1 through ultrafiltration membrane separation, the remaining components are called natto component F2 solution, i.e., a solution of components with molecular weights less than 26,000 Da and a solution of components with molecular weights greater than 29,000 Da.

[0025] S2: The keyword "antithrombotic" is searched for in the BIOPEP database using the "Bioactive peptides" module, and the characteristics of active peptides with antithrombotic function are analyzed, including basic properties such as molecular weight distribution, type of N-terminal amino acid residue, type of C-terminal amino acid residue, and net charge. In the present invention, the characteristic information is specifically as follows: a molecular weight of less than 2000 Da, the type of N-terminal amino acid residue being glycine G, the type of C-terminal amino acid residue being lysine K or arginine R, and a net charge of ±2 are considered to be characteristics of a peptide segment with potential antithrombotic activity.

[0026] S3: After obtaining the nattokinase component F1, the remaining natto component F2 is digested with a digestive enzyme such as trypsin for 4 hours to obtain a first natto enzymatic hydrolyzate. The first natto enzymatic hydrolyzate is subjected to peptide segment analysis using LC-MS / MS and de novo sequencing. Based on the above characteristics, the present invention achieves that some or all of the N-terminal amino acid residues in the obtained oligopeptides are glycine and the C-terminal amino acid residues are lysine K or arginine R. The C-terminal amino acid residues are lysine K or arginine R by cleaving the lysine carboxyl-terminal peptide bond or arginine carboxyl-terminal peptide bond as the target enzymatic cleavage site. For example, enzymatic cleavage using trypsin can yield oligopeptides whose C-terminal amino acid residues are lysine K or arginine R.

[0027] To obtain an oligopeptide whose N-terminal amino acid residue is glycine, it is necessary to count the types and quantities of amino acid residues preceding the glycine. However, considering the influence of enzymatic cleavage by a multiprotease, for example, a single multiprotease can achieve enzymatic cleavage at multiple sites, making it difficult to achieve enzymatic cleavage of all peptide bonds between glycine and all types of amino acids preceding the glycine. In the present invention, the single protease with the highest number of counts or the top two protease with the highest number of counts can be selected for enzymatic cleavage, thereby enabling an effective number of oligopeptides whose N-terminal amino acid residue is glycine to be obtained while also reducing the difficulty of designing a multiprotease for enzymatic cleavage.

[0028] The present invention further involves statistically determining the type and quantity of amino acid residues located before the arginine and designing a composite protease for the peptide bond between the amino acid before the arginine and arginine, simultaneously with the enzymatic cleavage described above, to obtain an effective number of oligopeptides whose N-terminal amino acid residue is arginine. Based on the characteristics described in step S2, the present invention does not require oligopeptides whose N-terminal amino acid residue is arginine R. However, according to the above-described content of the present invention, enzymatic cleavage is performed to obtain oligopeptides whose C-terminal amino acid residue is arginine R. Once an oligopeptide whose N-terminal amino acid residue is arginine is obtained, enzymatic cleavage is performed on this portion of the oligopeptide to obtain an oligopeptide whose C-terminal amino acid residue is arginine R. This allows the oligopeptide whose N-terminal amino acid residue is arginine to form free L-arginine. By increasing the amount of free L-arginine in the product, the present invention achieves the effects of dilating blood vessels and lowering blood pressure. Therefore, the present invention involves statistically determining the type and quantity of amino acid residues located before the arginine, and then performing enzymatic cleavage.

[0029] Based on the above statistics, the enzyme cleavage site is determined, and a composite protease combination is designed. Based on the appropriate enzymatic digestion conditions for the composite protease combination, natto component F2 is enzymatically digested to obtain a second enzymatic digestion solution. The composite protease of the present invention is required to achieve the enzymatic digestion objectives with the above characteristics and to obtain free L-arginine. The present invention achieves these objectives by using a combination of trypsin and chymotrypsin.

[0030] S4: The second enzymatic hydrolyzed solution is separated using a nanofiltration membrane with a molecular weight cut-off (MWCO) of 2000 Da. The second enzymatic hydrolyzed solution passes through the nanofiltration membrane, and the permeate is collected to obtain a solution of natto enzymatic hydrolyzed solution component F3.

[0031] S5: The solution of component F3 of the enzymatic natto decomposition liquid is further separated and purified by ion exchange chromatography to obtain a solution of component F4 of the enzymatic natto decomposition liquid having a net charge of ±2. The solution of component F4 of the enzymatic natto decomposition liquid thus obtained has the effects of thrombolysis and hypotensive action.

[0032] S6: The solution of natto enzyme hydrolyzed liquid component F4 and the solution of nattokinase component F1 obtained in step S1, which has certain effects such as anti-platelet aggregation, extracorporeal thrombolysis, hypotensiveness, and hypolipidemicness, are thoroughly mixed and then freeze-dried to obtain natto oligopeptide powder.

[0033] As described above, in the first aspect of the present invention, the nattokinase component F1 is first isolated to avoid destruction of nattokinase in the subsequent enzymatic hydrolysis process, and the natto enzymatic hydrolyzed solution component F4 having thrombolytic and antihypertensive effects is prepared. The nattokinase component F1 and the natto enzymatic hydrolyzed solution component F4 are then mixed to further enhance the therapeutic effect of the product and the amount of the product that can be used for treatment.

[0034] As described above, in the second aspect, the present invention analyzes and statistically analyzes the peptide segments of natto component F2, and designs a composite protease preparation to perform enzymatic cleavage using the amino acid preceding glycine, the amino acid preceding arginine, the carboxyl terminal of lysine, and the carboxyl terminal of arginine as target enzymatic cleavage sites to obtain natto enzymatic hydrolyzed liquid component F3, which contains a high content of free L-arginine with antihypertensive effect, and simultaneously increases the content of peptide segments with characteristic N-terminal and C-terminal amino acid residue types of antithrombotic peptide segments.

[0035] As described above, in the third aspect, the present invention further purifies component F4 of the enzymatic hydrolysis solution of natto, which has a molecular weight of 2000 Da or less, a net charge of ±2, and potential antithrombotic activity, by ultrafiltration membrane separation and ion exchange chromatography. Since arginine has one positive charge, it can be eluted together with component F4 in ion exchange chromatography. Therefore, the L-arginine obtained by the enzymatic hydrolysis of the present invention does not pose a need for improvement in the separation and purification process.

[0036] Example 1: A method for producing an oligopeptide, comprising the steps of:

[0037] S1: 100g of freeze-dried natto powder produced by fermenting soybeans with Bacillus subtilis natto was dissolved in 1000ml of saline. The freeze-dried natto powder solution was then passed through an ultrafiltration membrane with a molecular weight cutoff of 29,000 Da, and the permeate was collected to obtain components with a molecular weight of less than 29,000 Da. The permeate was then passed through an ultrafiltration membrane with a molecular weight cutoff of 26,000 Da, and the cutoff solution was collected to obtain components with a molecular weight greater than 26,000 Da but less than 29,000 Da, which was designated as nattokinase component F1. The cutoff solution from the first ultrafiltration and the permeate from the second ultrafiltration were combined to obtain natto component F2.

[0038] S2: The solution of natto component F2 is hydrolyzed with digestive enzymes such as trypsin for 4 hours to obtain natto enzyme hydrolyzed solution (enzyme hydrolysis pH 8.5, enzymatic hydrolysis temperature 50°C). The purpose of the enzymatic hydrolysis is to use the digestive enzymes to break down proteins into small peptides (small molecular peptides, usually consisting of 2 to 20 amino acid residues), and the enzyme hydrolyzed solution is subjected to peptide segment analysis using LC-MS / MS and de novo sequencing. The analytical conditions are as follows:

[0039] Chromatographic column: C18, 3 μm, 100 Å, 75 μm x 15 cm; Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in 80% acetonitrile / H2O; Chromatographic gradient: Spray voltage: 2.0 kV; Capillary temperature: 320 °C; RF lens: 40; Resolution settings: Level 1 is 120,000 @ m / z 200, Level 2 is 30,000 @ m / z 200; Mother ion scan range: m / z 350-1550; Child ion scan range: start from m / z 110; Fragmentation method: HCD (High Energy Collision Dissociation).

[0040] The raw data collected by mass spectrometry acquisition mode is used to perform de novo sequencing analysis using the de novo data analysis software PEAKS. The software parameters are set as follows:

[0041] Enzyme: No specificity, Variable modification: Oxidation (M), Deamidated (N, Q), Peptide mass tolerance: ±10 ppm, Fragment mass tolerance: 0.02 Da.

[0042] The types and quantities of amino acid residues preceding glycine and those preceding arginine were statistically analyzed. The distribution of the types and quantities of amino acid residues preceding glycine is shown in Figure 1, and the distribution of the types and quantities of amino acid residues preceding arginine is shown in Figure 2. The natto component F2 was enzymatically hydrolyzed to obtain a second enzymatic hydrolyzate using the tyrosine-glycine peptide bond, the phenylalanine-glycine peptide bond, the tyrosine-arginine peptide bond, the lysine carboxyl-terminal peptide bond, and the arginine carboxyl-terminal peptide bond as target enzymatic cleavage sites. The enzymatic hydrolysis conditions were as follows: trypsin:chymotrypsin = 1:(1-3), enzyme addition amount: 1%-3%, enzymatic hydrolysis temperature: 37°C-45°C, enzymatic hydrolysis pH: 7.5-8.5, and enzymatic hydrolysis time: 3-6 hours.

[0043] S3: The second enzymatic hydrolyzed solution is separated using a nanofiltration membrane with a molecular weight cutoff of 2000 Da, and the permeate is collected to obtain natto enzymatic hydrolyzed solution component F3, i.e., oligopeptides with a molecular weight of less than 2000 Da.

[0044] S4: An SP Sepharose High Performance strong cation exchange column is used, equilibrated with 20 mmol / L PB (pH 6.0). After loading the sample and re-equilibrating, elution is performed with a 0-1 M NaCl gradient over 0-40 min. The eluent stored for 11-19 min is collected and designated as natto enzyme hydrolyzed solution component F4.

[0045] S5: The natto enzyme hydrolyzed liquid component F4 and the nattokinase component F1 are thoroughly mixed and then freeze-dried to obtain natto oligopeptide powder.

[0046] Experimental Example 1: L-arginine content measurement

[0047] The freeze-dried natto powder raw material used in step S1 of Example 1 and the natto oligopeptide powder obtained in step S5 were each prepared into aqueous solution samples at a concentration of 100 μg / ml, and standard L-arginine was prepared in deionized water to prepare standard solutions at concentrations of 0.625, 1.25, 2.5, and 5.0 μmol / ml. 10 μL of each sample and standard were added to 20 μL of AQC (6-Aminoquinolyl-N-hydroxysuccinimidylcarbamate) solution for derivatization, and the free L-arginine content of each sample and standard was analyzed by high-performance liquid chromatography (HPLC) with fluorescence detection. The free L-arginine content in the freeze-dried natto powder sample was 0.23 g / 100 g, while the free L-arginine content in the natto oligopeptide powder sample was 5.5 g / 100 g. This indicates that the enzyme cleavage of the peptide bond before and after arginine in the freeze-dried natto powder protein is effective, and the free L-arginine obtained by enzymatic cleavage elutes together with the antithrombotic peptide components on a strong cation exchange column. Compared to the raw freeze-dried natto powder, the free L-arginine content in the natto oligopeptide powder was significantly improved.

[0048] Experimental Example 2: Peptide segment analysis

[0049] The peptide segments in the natto oligopeptide powder were sequenced using the same measurement method as in step S2 of Example 1, and the ratios of the types of N-terminal amino acid residues to the types of C-terminal amino acid residues were statistically determined. As a result, as shown in Figures 3 and 4, the types of N-terminal amino acid residues in the natto oligopeptide powder were mainly glycine, and the types of C-terminal amino acid residues were mainly lysine and some arginine, tyrosine, and tryptophan, which is consistent with the target enzymatic hydrolysis effect.

[0050] Experimental Example 3: Chronic thrombosis model in spontaneously hypertensive rats (SHR)

[0051] Freeze-dried natto powder and natto oligopeptide powder were mixed with deionized water to prepare a 1 g / ml paste. Eight WKY rats served as the control group, and 24 SHR rats were randomly divided into three groups (8 rats per group): the model group, the natto freeze-dried powder group (10 g / kg·d), and the high-dose natto oligopeptide powder group (10 g / kg·d). After anesthetizing the SHR rats with intraperitoneal injection of 3% pentobarbital sodium (2 mL / kg), the rats were inverted and a longitudinal incision of approximately 2 cm was made along the midline to isolate the bilateral cervical aortas. A homemade plastic mat was placed under the cervical aorta to protect the perivascular tissue. A small piece of filter paper saturated with 2 μL of 50% FeCl3 solution was placed in a circular pattern around the artery. The interface was gently pinched with tweezers to allow the filter paper to adhere to the arterial wall for 50 minutes, after which it was removed. This formed a non-occlusive arterial thrombus, occupying approximately 1 / 3 to 1 / 2 of the vascular space. The local tissue was then washed with saline. An appropriate amount of penicillin was sprayed into the wound to prevent infection, and the wound was then sutured layer by layer and returned to the cage. In the sham-operated group, a small piece of filter paper saturated with 2 μL of saline was placed in a circular pattern around the artery. After 50 minutes, it was removed and the wound was sutured. Starting the following day, the treatment group received the above-mentioned doses intragastrically, while the control and model groups received an equal volume of saline for one week. Blood pressure was measured in each group using a sphygmomanometer on days 1, 3, 5, and 7 after drug administration. Five measurements per animal were taken. SBP values ​​were recorded and averaged. On the 8th day, the animals are anesthetized with 20% urethane, and the blood vessels at the site of cervical aortic thrombosis are removed and placed on filter paper to absorb the remaining blood, and the mass is weighed using an electronic analytical balance.

[0052] As shown in Table 1, the SBP values ​​of spontaneously hypertensive rats (SHRs) were significantly higher than those of the control group, indicating that the formation of non-occlusive cervical aortic thrombi caused higher-than-normal blood pressure in SHRs. Compared to the model group, no significant differences were observed in SHRs receiving intragastric administration of natto freeze-dried powder. However, within one week of administration, SHRs receiving intragastric administration of natto oligopeptide powder showed a gradual decrease in blood pressure, demonstrating a significant difference. This demonstrates the excellent vasorelaxation and blood pressure-lowering effects of natto oligopeptide powder. The cervical aortic thrombus mass of SHRs in each group was also measured. Both the natto freeze-dried powder and natto oligopeptide powder groups had significantly lower thrombus mass than the model group (p<0.01). In the natto oligopeptide powder intervention group, the antiplatelet aggregation and anticoagulation effects of the antithrombotic peptide component F4, in addition to the fibrinolytic effect of nattokinase component F1, reduced further thrombus formation caused by endothelial cell damage, and therefore the antithrombotic effect obtained was even better than that of natto freeze-dried powder (p<0.01).

[0053] [Table 1]

[0054] [Table 2]

[0055] The present invention discloses a method for preparing natto oligopeptides and their application in thrombolysis and blood pressure reduction. This method uses natto as a raw material, first separating the nattokinase component by ultrafiltration, then sequencing the peptide segments of the remaining components and calculating the proportions of all amino acid residues located at the N-terminus of arginine. A complex protease preparation for this site is designed to perform enzymatic hydrolysis on the remaining components, resulting in a high L-arginine content in the hydrolysate, while also releasing natto antithrombotic peptides and ACE inhibitory peptides. The nattokinase component and the hydrolysate are then mixed and lyophilized to obtain natto oligopeptide powder. This preparation method preserves the thrombolytic activity of nattokinase in natto and enhances the thrombolytic effect of the final product, natto oligopeptides. Combined with the vasoregulatory effect of L-arginine, this method has great potential for applications in thrombolysis, blood pressure reduction, and other thrombotic cardiovascular diseases.

[0056] Experimental Example 4: Antithrombotic effect of natto enzyme hydrolyzed solution component F4

[0057] Thirty ICR male mice were randomly divided into a blank control group, a model group, and an antithrombosis group, each consisting of 10 mice. The blank control group received daily intraperitoneal injections of saline solution (0.01 mL / g body weight), the model group received intraperitoneal injections of 0.20% carrageenan in saline solution (0.01 mL / g body weight), and the antithrombosis group received oral administration of natto enzyme hydrolyzed solution component F4 (20 mg / g body weight) at the same time as the same dose of carrageenan was injected. After 10 days, the length of the black tails of the mice in each group was observed and recorded, and blood was collected and four blood coagulation parameters were detected using a semi-automated blood coagulation meter. The results, as shown in Table 3, show that mice in the model group developed black thrombi in their tails, but the tail thrombi length of mice in the natto enzyme hydrolyzed liquid component F4 intervention group was significantly shorter than that of the model group, and at the same time, the four coagulation indexes showed significant improvement compared to the model group, demonstrating that natto enzyme hydrolyzed liquid component F4 has a certain antithrombotic activity. The results of thrombus length and the four coagulation indexes for mice in each group (n=10) are shown in Table 3.

[0058] [Table 3]

[0059] *P<0.01 compared with the model group.

[0060] The method for producing natto oligopeptides of the present invention uses natto as a raw material, first separating the nattokinase component by ultrafiltration, then sequencing the peptide segments of the remaining components, and statistically determining the proportions of all amino acid residues located at the N-terminus of arginine. A multi-protease preparation is then designed for this site to perform enzymatic hydrolysis on the remaining components, resulting in a high L-arginine content in the hydrolysate, while also releasing natto antithrombotic peptides and ACE inhibitory peptides. The nattokinase component and the hydrolysate are then mixed and lyophilized to obtain natto oligopeptide powder. This production method retains the thrombolytic activity of nattokinase in natto and enhances the thrombolytic effect of the final product, natto oligopeptides. Combined with the vasoregulatory effect of L-arginine, this method has great potential for applications in thrombolysis, blood pressure reduction, and other thrombolytic cardiovascular diseases.

[0061] Finally, it should be noted that the above are only some specific examples of the present invention, and all derivatives that those skilled in the art can directly derive or associate with the contents disclosed in the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for producing natto antithrombotic oligopeptides, Searching a biologically active peptide database to determine the characteristics of the natto antithrombotic function-related peptide, the determined characteristic information including: a molecular weight of less than 2000 Da, a type of N-terminal amino acid residue being glycine G, a type of C-terminal amino acid residue being lysine K or arginine R, and a net charge of ±2; Preparing a natto peptide solution containing a nattokinase component solution and a natto small peptide solution; Sequencing the peptides in the natto small peptide solution, and based on the sequencing, collecting statistics on the type and number of amino acid residues located in front of glycine G and the type and number of amino acid residues located in front of arginine R of each peptide in the natto small peptide solution; ranking the samples based on statistical quantities, and then, based on the ranking, using a composite protease to cleave the tyrosine-glycine peptide bond, the phenylalanine-glycine peptide bond, the tyrosine-arginine peptide bond, the lysine carboxyl terminal peptide bond, and the arginine carboxyl terminal peptide bond as enzymatic cleavage sites, the composite protease being composed of trypsin and chymotrypsin, with a trypsin:chymotrypsin ratio of 1:(1-3), at an enzymatic decomposition temperature of 37°C to 45°C, a pH of 7.5 to 8.5, and a time of 3 to 6 hours; filtering the solution of cleaved peptides and collecting a solution of peptides having a molecular weight of less than 2000 Da; Separating and purifying a solution of peptides having a molecular weight of less than 2000 Da, and collecting a solution of peptides having a net charge of ±2 to obtain a first oligopeptide; mixing the solution of the peptide with a net charge of ±2 with a solution of a nattokinase component to obtain a second oligopeptide; Including, preparing the nattokinase component solution includes: passing the natto powder solution through an ultrafiltration membrane having a molecular weight cutoff of 29,000 Da to obtain a first permeate; and passing the first permeate through an ultrafiltration membrane having a molecular weight cutoff of 26,000 Da to obtain a second permeate and a second cutoff liquid, wherein the second cutoff liquid is the nattokinase component solution; preparing the natto small peptide solution includes adding a digestive enzyme to the first permeate solution and the second blocking solution to obtain the natto small peptide solution, wherein the digestive enzyme is trypsin; Ranking based on statistical quantities is ranking different types of pre-glycine G amino acid residues by number; Ranking different types of amino acid residues preceding arginine R by number; based on the ranking of the number of amino acid residues preceding the different types of glycine G, cleaving the peptide bond between the type of amino acid residue with the largest number or the type with the first number ranking and glycine G to obtain a peptide whose N-terminal amino acid residue type is glycine G; cleaving the peptide bond between the type of amino acid residue with the largest number or the type with the first number ranking and arginine R based on the ranking of the number of amino acid residues preceding different types of arginine R to obtain a peptide whose N-terminal amino acid residue type is arginine R; cleaving the lysine carboxyl-terminal peptide bond to obtain a peptide in which the type of C-terminal amino acid residue is lysine K; cleaving the arginine carboxyl-terminal peptide bond of a peptide whose non-N-terminal amino acid residue is arginine R to obtain a C-terminal amino acid residue whose type is arginine R; and cleaving the arginine R of a peptide whose N-terminal amino acid residue is arginine R to obtain free L-arginine; Including, 1. A method for producing natto antithrombotic oligopeptides, comprising:

2. filtering the solution of cleaved peptides includes filtering the solution of cleaved peptides through a nanofiltration membrane having a molecular weight cutoff of 2000 Da, and the permeate of the nanofiltration membrane is a solution of peptides having a molecular weight of less than 2000 Da; Separating and purifying a solution of peptides having a molecular weight of less than 2000 Da includes separating and purifying a solution of peptides having a molecular weight of less than 2000 Da by ion exchange chromatography; The method for producing natto antithrombotic oligopeptide according to claim 1.

3. The ion exchange chromatography comprises a strong cation exchange column. The method for producing natto antithrombotic oligopeptide according to claim 2.

4. The ion exchange chromatography is carried out using an SP Sepharose High Performance strong cation exchange column. After loading the sample, the column is equilibrated with 20 mmol / L PB, and eluted with a 0-1 M NaCl gradient from 0 to 40 min. The eluate is collected from the retention time of 11 to 19 min to obtain a solution of peptides with a net charge of ±2. The method for producing natto antithrombotic oligopeptide according to claim 3.

5. The natto is produced from soybeans. The method for producing natto antithrombotic oligopeptide according to claim 1.

6. Further comprising freeze-drying the first oligopeptide to obtain natto oligopeptide powder. The method for producing natto antithrombotic oligopeptide according to claim 1.

7. Further comprising freeze-drying the second oligopeptide to obtain natto oligopeptide powder. The method for producing natto antithrombotic oligopeptide according to claim 1.

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