Preparation method of equine myocardial peptide with antioxidant and antithrombotic effects

Abstract

This invention relates to the fields of enzyme engineering and bioactive peptide preparation technology, and discloses a method for preparing equine cardiac muscle peptides with antioxidant and antithrombotic effects, comprising the following steps: S1: Pre-treating equine heart tissue to obtain equine cardiac muscle plasma; S2: Compound enzymatic hydrolysis and directing agent regulation: Adding a compound enzyme system and an antithrombotic directing agent to the equine cardiac muscle plasma; S3: Affinity chromatography enrichment. This invention constructs a synergistic process of compound enzymatic hydrolysis, directing regulation, and affinity enrichment, specifically addressing the technical problems of incomplete tissue fragmentation, low enzymatic hydrolysis efficiency, insufficient yield of active peptides, and easy oxidative inactivation in equine cardiac muscle due to the high proportion of type IIa myofibrous fibers and dense connective tissue. Furthermore, it employs a specific ratio of compound enzyme system adapted to the characteristics of equine cardiac muscle, significantly improving enzymatic hydrolysis efficiency; introduces a cationic directing agent to directionally increase the proportion of antithrombotic peptides generated; and combines thrombin affinity chromatography to achieve efficient enrichment of target peptides, significantly enhancing antithrombotic function while ensuring high antioxidant activity.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and bioactive peptide preparation technology, and in particular to a method for preparing horse cardiac muscle peptides with antioxidant and antithrombotic effects. Background Technology

[0002] Cardiac peptides are bioactive peptides extracted from animal myocardial tissue. They have multiple physiological functions such as antioxidation, anti-inflammation, and cardiovascular protection, and are widely used in the food, health products, and cosmetics industries. Currently, related research and patented technologies mainly focus on the preparation of cardiac peptides from common livestock such as pigs, cattle, and sheep. The processes mainly employ methods such as mechanical crushing, high-temperature sterilization, and stepwise enzymatic hydrolysis, focusing on improving single indicators such as peptide yield, purity, and antioxidant activity, thus forming a relatively mature production system.

[0003] It is worth noting that animal myocardial tissue naturally contains precursor proteins with sequences such as RGD (arginine, glycine, and aspartic acid) and KGD (lysine, glycine, and aspartic acid). These sequences are the core functional units of antithrombotic active peptides. In equine myocardium, proteins such as troponin T and myosin light chain in type IIa myocardial fibers have a significantly higher proportion of RGD / KGD related motifs in their amino acid sequences than those in pig, cattle, and sheep myocardium (approximately 12% to 15% higher), thus naturally possessing the material basis for preparing antithrombotic active peptides.

[0004] However, existing technologies have not addressed the preparation of equine cardiac peptides, nor have they considered the impact of structural and biochemical differences between different myocardial tissues on process adaptability. Equine myocardium possesses characteristics significantly different from those of pig, bovine, and sheep myocardium: it is predominantly composed of type IIa myofibrous fibers (accounting for 58%–70%), has a high proportion of connective tissue (approximately 12%), and exhibits strong and specific activity of endogenous proteases (such as citrate synthase and 3-hydroxyacyl-CoA dehydrogenase). These characteristics result in its strong tissue toughness, difficulty in enzymatic hydrolysis, and easy oxidative breakage of peptide chains. For example, patent CN108607091B discloses a formula for a blood pressure-lowering polypeptide herbal solid beverage and cardiac peptides. The powder preparation method involves the preparation process of bovine and ovine cardiac muscle peptides. The enzymatic hydrolysis system and process parameters are designed for the characteristics of bovine and ovine cardiac muscle tissue, but are not adapted to the specific characteristics of equine cardiac muscle. Furthermore, it does not target the enzymatic hydrolysis regulation of the naturally contained antithrombotic precursor proteins. If the existing process is directly applied to treat equine cardiac muscle, it will face problems such as incomplete tissue disruption, low enzymatic hydrolysis efficiency, insufficient yield of target active peptides, and easy oxidation and inactivation. Not only will it be difficult to achieve efficient industrial production, but it will also be unable to fully release the natural antithrombotic potential of equine cardiac muscle, and will not meet the dual functional requirements of equine cardiac muscle peptides having both high antioxidant and significant antithrombotic activity. Summary of the Invention

[0005] The technical problem to be solved by this invention is that the existing technology is not adapted to the special structure of horse myocardium, resulting in low enzymatic hydrolysis efficiency and peptide yield, and it is unable to synergistically enrich peptides with dual antioxidant and antithrombotic activities. Therefore, we propose a method for preparing horse myocardial peptides with antioxidant and antithrombotic effects.

[0006] To achieve the above objectives, this application adopts the following technical solution: a method for preparing horse cardiac muscle peptides with antioxidant and antithrombotic effects, comprising the following steps:

[0007] S1: Horse myocardial plasma was prepared by pretreatment of horse heart tissue;

[0008] S2: Complex Enzymatic Hydrolysis and Directing Agent Regulation: A complex enzyme system and an antithrombotic directing agent are added to the horse myocardial plasma, and the enzymatic hydrolysis reaction is carried out at a pH of 7.3-7.7 and a temperature of 48-52℃. The complex enzyme system consists of alkaline protease, activated pancreas, and neutral protease. The amount of activated pancreas added is 12-16% of the total mass of the horse myocardial plasma, and the amount of the antithrombotic directing agent added is 0.2-0.4% of the total mass of the horse myocardial plasma.

[0009] S3: Affinity chromatography enrichment: The enzymatic hydrolysate from step S2 is subjected to solid-liquid separation, and the resulting supernatant is purified by thrombin affinity chromatography column to collect the fraction rich in antithrombotic peptides.

[0010] S4: Dry the components obtained in step S3 to obtain the horse myocardial peptide powder.

[0011] Preferably, in step S2, the amount of alkaline protease added is 0.5-0.7% of the total mass of horse myocardial plasma, and the amount of neutral protease added is 0.08-0.12% of the total mass of horse myocardial plasma.

[0012] Preferably, in step S2, the antithrombotic directing agent is chitosan quaternary ammonium salt.

[0013] Preferably, in step S2, the amount of activated pancreas added is 14% of the total mass of horse myocardial plasma, and the amount of antithrombotic directing agent added is 0.3% of the total mass of horse myocardial plasma.

[0014] Preferably, in step S2, the pH value of the enzymatic hydrolysis reaction is 7.5.

[0015] Preferably, in step S2, during the enzymatic hydrolysis reaction, sodium ascorbate is added to the reaction system every 2 hours, and the amount added is 0.05% of the current total mass of the reaction system.

[0016] Preferably, in step S3, the elution process of the thrombin affinity chromatography column is performed by gradient elution using a Tris-HCl buffer containing 0.5 mol / L NaCl and with a pH of 7.4.

[0017] Preferably, in step S1, the pretreatment includes: cutting the horse heart tissue into blocks, freezing and thawing them, and then sequentially performing ultrasonic disruption and mechanical shredding at a frequency of 40 kHz to obtain horse myocardial plasma with a particle size ≤ 500 μm.

[0018] Preferably, in step S4, the drying specifically involves: filtering and vacuum concentrating the components obtained in step S3 until the solid content is 18-22%, and then freeze-drying them.

[0019] Furthermore, the present invention also relates to an embodiment, specifically a horse cardiac muscle peptide with antioxidant and antithrombotic effects, which satisfies the following: half-maximal scavenging capacity (EC50) for DPPH free radicals. 50 ≤0.00267mg / L; thrombin inhibition rate ≥64.7%; peptides with a molecular weight less than 1000Da account for no less than 92% of the total peptide mass.

[0020] The technical effects and advantages of this invention are as follows:

[0021] This invention addresses the technical problems of incomplete tissue fragmentation, low enzymatic hydrolysis efficiency, insufficient yield of active peptides, and easy oxidative inactivation in equine myocardium due to the high proportion of type IIa myofiber and dense connective tissue. By constructing a synergistic process of complex enzymatic hydrolysis, targeted regulation, and affinity enrichment, this invention specifically solves these problems. The process employs a specific ratio of alkaline protease, activated pancreas, and neutral protease in a complex enzyme system adapted to the protein composition and endogenous protease distribution characteristics of equine myocardium, significantly improving enzymatic hydrolysis efficiency. Simultaneously, a cationic targeting agent is introduced to regulate the exposure of enzymatic hydrolysis sites through electrostatic interactions, resulting in the presence of RG in the product. The D / KGD functional sequence significantly increases the proportion of antithrombotic peptides generated. Thrombin affinity chromatography then efficiently enriches the target peptides, avoiding interference from other peptides. This method ensures high antioxidant activity while significantly enhancing antithrombotic function, ultimately achieving a technological breakthrough in the synergistic preparation of peptides with both high antioxidant and antithrombotic activity from equine myocardium. This not only fills the gap in equine myocardial peptide products and dedicated preparation processes but also overcomes the limitations of existing technologies that focus only on a single function, have poor compatibility with equine myocardium, and have low yields. It provides a feasible path for the high-value utilization of equine myocardial resources. Attached Figure Description

[0022] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings, the same reference numerals are used to refer to the same parts:

[0023] Figure 1 This is a schematic diagram of the process flow of the present invention. Detailed Implementation

[0024] It is readily understood that, based on the technical solution of this invention, those skilled in the art can propose various interchangeable structural methods and implementations without altering the essential spirit of the invention. Therefore, the following detailed embodiments and accompanying drawings are merely illustrative examples of the technical solution of this invention and should not be considered as the entirety of the invention or as limitations or restrictions on the technical solution of this invention.

[0025] Reference Figure 1 As shown, the present invention provides a technical solution: a method for preparing equine cardiac peptides with antioxidant and antithrombotic effects:

[0026] Raw materials and reagents include:

[0027] Fresh horse heart: Collected within 2 hours after slaughter at a large-scale livestock base, washed and cleaned to remove fascia, blood vessels and other connective tissues, quickly cut into 5cm×5cm pieces, pre-frozen at -15℃ for 6 hours for later use, to ensure that the muscle fiber structure has not been irreversibly damaged.

[0028] Enzyme preparations: alkaline protease (enzyme activity 200,000 U / g, Michaelis constant Km=0.8mmol / L), activated pancreas (commercially available pancreatic enzyme preparation, enzyme activity 150,000 U / g, activated at 4℃ for 24h), and neutral protease (enzyme activity 180,000 U / g, optimal pH 7.0-7.5), all of which are food-grade high-purity enzymes.

[0029] The antithrombotic targeting agent, hereinafter referred to as the targeting agent, is a chitosan quaternary ammonium salt with a molecular weight of 5000 Da and a degree of substitution ≥80%. Its structural integrity has been verified by infrared spectroscopy. It utilizes its cationic properties to interact with the electrostatic regions of specific proteins, affecting the accessibility of enzymatic hydrolysis sites, thereby indirectly regulating the release pattern of peptides containing functional sequences.

[0030] Thrombin affinity medium: Epoxy activated agarose gel-thrombin conjugate (coupling efficiency ≥90%, column capacity ≥20mg / mL).

[0031] Other reagents: Sodium ascorbate (food grade), potassium dihydrogen phosphate, disodium hydrogen phosphate (analytical grade, used for preparing buffer solutions), DPPH reagent (purity ≥98%), ABTS reagent (purity ≥97%), thrombin (bovine plasma source, activity ≥20U / mg), ADP (platelet aggregation inducer, purity ≥99%), bovine serum albumin (standard), etc.

[0032] Process steps:

[0033] Based on the characteristics of equine myocardial tissue (58%–70% type IIa myofibrous tissue and 12% connective tissue), the following basic process was designed to prepare equine myocardial peptides with both antioxidant and antithrombotic activities through the synergistic effects of complex enzymatic hydrolysis, targeted agent regulation, and affinity chromatography enrichment. The key parameters are set as follows:

[0034] Raw material pretreatment: Take horse hearts thawed to -2℃~0℃, rinse 3 times with phosphate buffer containing 0.02% sodium ascorbate (pH 7.2, concentration 0.05mol / L), 10min each time, cut into 1cm³ pieces, pre-freeze at -15℃ for 6h, thaw to a core temperature of 4℃~6℃, sonicate at 40kHz for 15-25min, grind into a paste with a particle size ≤500μm using a colloid mill, add deionized water at a material-to-liquid ratio of 1:3 (w / v), and stir at 300rpm for 30min to obtain horse myocardial plasma.

[0035] Sterilization treatment: Moist heat sterilization at 100-110℃ for 12-18 minutes to destroy the activity of endogenous proteases and moderately denature muscle proteins to facilitate enzymatic hydrolysis, followed by rapid cooling to 50℃ at a rate ≥2℃ / min.

[0036] Complex enzymatic hydrolysis and directing agent regulation: A complex enzyme system was added, including 0.5%-0.7% alkaline protease, 12%-16% activated pancreas, and 0.08%-0.12% neutral protease, all by mass ratio, based on the total mass of myocardial plasma. A directing agent was added simultaneously at a mass ratio of 0.2%-0.4%. The pH was adjusted to 7.3-7.7, and the enzyme was hydrolyzed at a constant temperature of 48-52℃ with stirring for 5-7 hours. 0.05% sodium ascorbate was added every 2 hours to maintain a reducing environment. It should be noted that the directing agent regulates protein conformation and the exposure of enzymatic hydrolysis sites through electrostatic interactions, directionally releasing peptides containing antithrombotic motifs from equine myocardium. This increases the proportion of peptides containing antithrombotic sequences in the hydrolysate, laying the foundation for subsequent affinity chromatography enrichment.

[0037] Enzyme inactivation: Incubate at 85-95℃ for 15-25 minutes.

[0038] Centrifugation: Centrifuge at 7000-9000 rpm for 20 min and collect the supernatant.

[0039] Affinity chromatography enrichment: The chromatography column was washed with equilibration buffer (Tris-HCl buffer at pH 7.4, 0.02 mol / L), the supernatant was loaded, and the column was washed with equilibration buffer until the absorbance was ≤0.02. The column was then eluted with a gradient of Tris-HCl buffer containing 0.5 mol / L LDENaCl at pH 7.4. The elution peak at 8-12 min was collected, which is the enriched fraction of the target active peptide. Because the directing agent has pre-regulated the peptide distribution of the enzymatic hydrolysis products, peptides containing antithrombotic functional sequences are more likely to specifically bind to the thrombin affinity medium, achieving a synergistic effect of directing regulation and precise enrichment.

[0040] Microfiltration and concentration drying: Filtered through a 0.45μm filter membrane, vacuum concentrated at -0.09MPa and 50℃ to a solid content of 18%-22%, then freeze-dried at -50℃ and 10Pa for 12 hours, resulting in a moisture content of ≤3% for the Demacocardial Peptide powder.

[0041] This invention also relates to an embodiment, specifically a horse cardiac muscle peptide with antioxidant and antithrombotic effects, which is a peptide mixture prepared by the above-described method, and simultaneously satisfies:

[0042] (a) The half-maximal scavenging capacity (EC50) for DPPH radicals is ≤0.00267 mg / L;

[0043] (b) Inhibition rate of thrombin ≥64.7%;

[0044] (c) Peptides with a molecular weight of less than 1000 Da account for no less than 92% of the total peptide mass.

[0045] To verify the rationality of the optimization range of key parameters in the process and the impact of different parameter combinations on product performance, and to clarify the feasibility of the process and the optimal parameter ratio, the following examples were set up. Each example only changes a single key parameter, and the remaining steps are consistent with the process.

[0046] Example 1: Preferred Example

[0047] This embodiment employs a reasonable combination of parameters determined through prior exploration in the process to achieve synergistic optimization of enzymatic hydrolysis efficiency, antioxidant activity, and antithrombotic activity. The specific steps are as follows:

[0048] Step 1: Raw material pretreatment: Take 1 kg of horse heart thawed to -2℃~0℃, rinse 3 times with phosphate buffer (pH 7.2, 0.05mol / L) containing 0.02% sodium ascorbate (pH 7.2, 0.05mol / L) for 10 min each time to thoroughly remove surface impurities and residual blood, cut into 1 cm³ uniform small pieces, pre-freeze at -15℃ for 6 h, and then thaw naturally to a core temperature of 4℃~6℃. Place in an ultrasonic device and continuously process at 40 kHz and 500W power for 20 min, stirring once every 5 min to ensure uniform ultrasonication. After ultrasonication, mechanically grind the horse heart 3 times using a colloid mill (8000 rpm) to obtain porridge-like horse heart porridge with a particle size ≤500μm. Add 3L of deionized water at a material-to-liquid ratio of 1:3 (w / v) and stir at 300 rpm for 30 min to prepare homogeneous horse heart porridge.

[0049] Step 2: Sterilization: Transfer the horse myocardial plasma into a constant temperature enzymatic hydrolysis tank, turn on the steam sterilization system, and sterilize at 105℃ for 15 minutes. After sterilization, cool it down to 50℃ at a rate of 2.5℃ / min through the jacket cooling system for later use.

[0050] Step 3: Synergistic Regulation of Complex Enzymatic Hydrolysis and Directing Agent: A complex enzyme system was added to the sterilized myocardial plasma, including 0.6% (mass ratio) of alkaline protease, 14% (mass ratio) of activated pancreas, and 0.1% (mass ratio) of neutral protease. Simultaneously, a directing agent (chitosan quaternary ammonium salt) was added at 0.3% (mass ratio). The pH of the system was adjusted to 7.5, and the constant temperature system was turned on to maintain 50°C. The stirring speed was 200 rpm, and the hydrolysis time was 6 hours. During this period, 0.05% sodium ascorbate (based on the current system mass) was added every 2 hours. The pH was maintained at ≤±0.1 through online pH compensation to ensure stable enzyme activity. The directing agent regulates the conformation of equine myocardial proteins through electrostatic interaction, making the enzymatic hydrolysis sites related to the antithrombotic functional sequence more easily recognized by the complex enzyme system, thereby improving the generation efficiency of the target peptide.

[0051] Step 4: Enzyme inactivation: After enzymatic hydrolysis, heat to 90℃ at a rate of 3℃ / min and hold for 20min to ensure that the residual enzyme activity is ≤0.1%.

[0052] Step 5: Centrifugation: Cool the enzymatic hydrolysate to below 25°C, transfer it to a high-speed refrigerated centrifuge, centrifuge at 8000 rpm for 20 min, collect the supernatant, and discard the bottom precipitate (mainly undegraded connective tissue and inactivated enzymes).

[0053] Step 6: Affinity Chromatography Enrichment: Thrombin affinity medium is loaded into the chromatography column. The column bed is washed with equilibration buffer (Tris-HCl buffer, pH 7.4, 0.02 mol / L) at a flow rate of 1.5 mL / min until the baseline is stable. The supernatant is injected into the chromatography column using a constant flow pump. After loading, the column is washed with equilibration buffer at a flow rate of 1.5 mL / min until the absorbance value of the UV detector (280 nm) is ≤0.02. The column is then switched to elution buffer (containing 0.5 mol / L NaCl, Tris-HCl buffer, pH 7.4) and eluted at a gradient flow rate of 1.0 mL / min. The elution peaks at 8-12 min are collected to obtain the antithrombotic peptide enrichment solution. Due to the structural characteristics of the target peptide generated by the directing agent, the binding specificity of the target peptide to the thrombin affinity medium is significantly higher than that of ordinary peptides, resulting in a significant improvement in enrichment efficiency.

[0054] Step 7: Microfiltration and Concentration Drying: The enriched solution was filtered through a 0.45 μm polyethersulfone filter membrane to remove trace suspended matter, and then transferred to a vacuum falling film concentrator to concentrate to a solid content of 20% at -0.09 MPa and 50 °C. Subsequently, the concentrate was transferred to a vacuum freeze dryer and dried at -50 °C and 10 Pa for 12 h to obtain white and loose horse myocardial peptide powder.

[0055] Example 2:

[0056] The only difference from Example 1 is that the proportion of activated pancreas added to the complex enzyme system in step 3 is adjusted to 12% (mass ratio, lower limit of process parameter range). All other process parameters are completely consistent with those in Example 1, which is used to verify the effectiveness of the process at the lower limit of the proportion of activated pancreas.

[0057] Example 3:

[0058] The only difference from Example 1 is that the proportion of activated pancreas added to the complex enzyme system in step 3 is adjusted to 16% (mass ratio, upper limit of process parameter range). All other process parameters are consistent with those in Example 1. This is to verify the rationality of the upper limit of the proportion of activated pancreas and to avoid excessive hydrolysis of peptides due to excessive enzyme content.

[0059] Example 4:

[0060] The only difference from Example 1 is that the amount of the directing agent added in step 3 is adjusted to 0.2% (mass ratio, lower limit of process parameter range), and the other process parameters are the same as in Example 1.

[0061] Example 5:

[0062] The only difference from Example 1 is that the amount of the directing agent added in step 3 is adjusted to 0.4% (mass ratio, upper limit of process parameter range), and the other process parameters are the same as in Example 1, verifying the saturation effect of the upper limit of the directing agent dosage.

[0063] Example 6:

[0064] The only difference from Example 1 is that the pH of the enzymatic hydrolysis system in step 3 is adjusted to 7.3 (the lower limit of the process parameter range), while the other process parameters are the same as in Example 1, to explore the effect of the lower limit of pH on the synergistic activity of the complex enzyme system.

[0065] Example 7:

[0066] The only difference from Example 1 is that the pH of the enzymatic hydrolysis system in step 3 is adjusted to 7.7 (the upper limit of the process parameter range), while the other process parameters remain the same as in Example 1, to verify the effect of the upper limit of pH on the enzymatic hydrolysis efficiency and product activity.

[0067] To clarify the technical effects of the addition of the directing agent, affinity chromatography enrichment, and optimization of key parameters in this process, the following comparative examples were set up. Each comparative example only changed the target variable, and the remaining process parameters were the same as in Example 1.

[0068] Comparative Example 1:

[0069] The difference from Example 1 is that no directing agent was added in step 3, and the affinity chromatography enrichment step in step 6 was omitted. This is to compare the core role of the directing agent and affinity chromatography in enhancing antithrombotic activity.

[0070] Comparative Example 2:

[0071] The difference from Example 1 is that step 6, affinity chromatography enrichment, is omitted, while the remaining process parameters are the same as in Example 1, verifying the key role of affinity chromatography in the enrichment of antithrombotic peptides.

[0072] Comparative Example 3:

[0073] The difference from Example 1 is that no directing agent was added in step 3, while the remaining process parameters were the same as in Example 1, thus clarifying the inducing effect of the directing agent in the directional generation of antithrombotic peptides.

[0074] Comparative Example 4:

[0075] The difference from Example 1 is that the proportion of activated pancreas added in step 3 is adjusted to 10% (by mass), while the other process parameters are the same as in Example 1.

[0076] Comparative Example 5:

[0077] The difference from Example 1 is that the proportion of activated pancreas added in step 3 is adjusted to 18% (by mass), while the other process parameters are the same as in Example 1.

[0078] Comparative Example 6:

[0079] The difference from Example 1 is that the amount of the directing agent added in step 3 is adjusted to 0.1% (by mass), while the remaining process parameters are the same as in Example 1, verifying the targeting effect defect caused by insufficient directing agent dosage.

[0080] Comparative Example 7:

[0081] The difference from Example 1 is that the amount of the guiding agent added in step 3 is adjusted to 0.5% (by mass), while the other process parameters are the same as in Example 1.

[0082] Comparative Example 8:

[0083] The difference from Example 1 is that the pH of the enzymatic hydrolysis system in step 3 is adjusted to 7.1, while the other process parameters are the same as in Example 1, verifying the inhibitory effect of pH below the range on the activity of the complex enzyme.

[0084] Comparative Example 9:

[0085] The difference from Example 1 is that the pH of the enzymatic hydrolysis system in step 3 is adjusted to 7.9, while the other process parameters are the same as in Example 1.

[0086] The products of Examples 1-7 and Comparative Examples 1-9 were systematically tested, including antioxidant activity, antithrombotic activity, and physicochemical properties. Three parallel samples were set up for each sample, and the test results were taken as the arithmetic mean to verify the range of process parameters and the technical effect of the process.

[0087] Test Example 1: DPPH Free Radical Scavenging Capacity Detection

[0088] The antioxidant activity of the samples was detected using the DPPH method at a wavelength of 517 nm, with glutathione as a positive control. The half-maximal scavenging capacity (EC50) was calculated. 50 The antioxidant capacity (AO) is calculated using the following formula: AO = EC 50 (S) / EC 50 (R), where: AO represents antioxidant capacity, EC 50 (S) represents the half-maximal clearance (MCL) of the peptide sample (mg / L), EC 50 (R) represents the half-maximal clearance (IC50) of glutathione (mg / L). The result is expressed as the arithmetic mean of parallel measurements, retained to three significant figures. The smaller the AO value, the stronger the antioxidant activity. The test results are shown in Table 1 below:

[0089]

[0090] Table 1

[0091] As shown in Table 1 above, the DPPH-EC50 values ​​of Examples 1-7 are all lower than those of Comparative Examples 1-9, and the AO values ​​are even lower, indicating that the samples within the process parameter range have better antioxidant activity. Among them, Example 1 has the lowest AO value and the strongest antioxidant activity, proving that the parameter combination of the preferred examples can maximize the maintenance of the peptide's oxidative resistance. The AO values ​​of Comparative Examples 4-9 and Comparative Examples 1-3 are all greater than 4.76, indicating that steps outside the parameter range of the preferred examples or missing some steps will lead to a decrease in antioxidant activity.

[0092] Test Example 2: ABTS Free Radical Scavenging Ability Test

[0093] The antioxidant activity of the samples was further verified using the ABTS method, with a detection wavelength of 734 nm. Glutathione was used as a positive control, and EC was calculated. 50 The antioxidant capacity (AO) is calculated using the following formula: AO = EC 50 (S) / EC 50 (R), where: AO represents antioxidant capacity, EC 50 (S) represents the half-maximal clearance (MCL) of the peptide sample (mg / L), EC 50 (R) represents the half-maximal clearance of glutathione (unit: mg / L). The results are expressed as the arithmetic mean of parallel measurements, retained to three significant figures. The test results are shown in Table 2 below:

[0094]

[0095] Table 2

[0096] The ABTS test results and DPPH test results provide complementary verification, and the ABTS-EC results in Examples 1-7 are further validated. 50 The values ​​were all lower than those of the comparative example, and the AO values ​​were between 2.19 and 2.32, which were better than those of the comparative example. The ABTS-AO value of Example 1 was the smallest, which further proves that the parameter combination of the preferred example can effectively retain the antioxidant activity of the peptide. The AO values ​​of the excessive directing agent in the comparative example 7 and the excessively high pH in the comparative example 9 were close to those of the comparative example 1, indicating that the parameters exceeding the scope of the present invention and the absence of some steps in the present invention have the same negative impact on antioxidant activity.

[0097] Test Example 3: Thrombin Inhibition Rate Detection

[0098] The inhibitory effect of samples on thrombin-catalyzed fibrinogen coagulation was detected using a coagulation analyzer. The reaction system contained thrombin (0.5 U / mL), fibrinogen (2 mg / mL), and samples of different concentrations. The thrombin inhibition rate (%) was calculated. The higher the inhibition rate, the stronger the antithrombotic activity. The test results are shown in Table 3 below.

[0099]

[0100] Table 3

[0101] The data in Table 3 above show that the thrombin inhibition rates of Examples 1-7 are significantly higher than those of Comparative Examples 1-9. Among them, Example 1 has the highest inhibition rate, which is 2.4 times higher than Comparative Example 1, 59% higher than Comparative Example 2, and 88% higher than Comparative Example 3. This proves that the synergistic design of targeted induction and affinity chromatography enrichment can efficiently enrich antithrombotic peptides. The inhibition rates of Examples 2-7 are between 64.7% and 69.2%, which are all better than all comparative examples, verifying the effectiveness of the parameter range of this scheme. The parameters of Comparative Examples 4-9 are outside the parameter range of this scheme, and their inhibition rates are only between 39.8% and 44.2%, indicating that parameters outside the range cannot achieve ideal antithrombotic activity.

[0102] Test Example 4: Platelet Aggregation Inhibition Rate Detection

[0103] Platelet aggregation analyzer was used with ADP as the inducer (final concentration 10 μmol / L) to detect the aggregation rate of rabbit platelet-rich plasma under the action of the sample, and the aggregation inhibition rate (%) was calculated to reflect the inhibitory effect of the sample on key steps of thrombosis. The test results are shown in Table 4 below:

[0104]

[0105] Table 4

[0106] As shown in Table 4 above, the platelet aggregation inhibition rate test results are consistent with the thrombin inhibition rate. The inhibition rates of Examples 1-7 range from 58.3% to 65.6%, with Example 1 achieving the highest rate of 65.6%, which is superior to the comparative examples. The inhibition rate of Comparative Example 1 is only 18.5%, indicating that the product has no significant anti-platelet aggregation activity when some steps of this scheme are omitted. The inhibition rates of Comparative Example 2 with only the directing agent and Comparative Example 3 with only chromatography are 42.3% and 35.7%, respectively, further illustrating that the synergistic effect of the directing agent and affinity chromatography is indispensable. The inhibition rates of Comparative Examples 4-9 are all below 40%, verifying the necessity of the parameter range of this scheme.

[0107] Test Example 5: Physicochemical Performance Testing

[0108] The molecular weight distribution of peptides was determined by GPC, and the peptide yield and purity were calculated by a combination of the Kjeldahl method and high-performance liquid chromatography. The impact of the process on raw material utilization and product quality was evaluated. The test results are shown in Table 5 below.

[0109]

[0110] Table 5

[0111] As shown in Table 5 above, the proportions of peptides below 1000 Da (92.3%-94.2%), peptides below 500 Da (82.9%-85.7%), and peptide purity (76.8%-77.5%) in Examples 1-7 are all higher than those in the comparative examples. Among them, Example 1 has the best performance in all indicators, proving that the parameter combination of this scheme can improve the enzymatic hydrolysis efficiency and product purity. The peptide yield of the examples is 19.8%-20.3%, which remains stable and meets the needs of industrial production. In Comparative Example 5, the proportion of peptides below 500 Da due to excessive activation of pancreas is only 78.3%, and the peptide purity is 75.2%, indicating that excessive enzymatic hydrolysis will lead to peptide structure destruction and decreased purity. The peptide yield of Comparative Example 3 without the directing agent is only 19.0%, indicating that the directing agent can improve the utilization rate of raw materials. In Example 5, the viscosity of the system slightly increased due to the excessive directing agent (0.4%), the utilization rate of raw materials slightly decreased, and the peptide yield was slightly lower than that of the preferred examples.

[0112] Test Example 6:

[0113] To visually present the performance differences of key parameters within and outside the optimization range, and to clarify the technical value of parameter optimization, the core parameters and their corresponding core performance indicators are summarized in Table 6 below:

[0114]

[0115] Table 6

[0116] Table 6 above shows the performance differences of key parameters under different values. Within the range of this scheme (12%-16% activated pancreas, 0.2%-0.4% directing agent, pH 7.3-7.7), the thrombin inhibition rate is ≥64.7%, the proportion of peptides below 500 Da is ≥82.9%, and the peptide purity is ≥76.8%, which is better than the values ​​outside the range. The performance indicators of the preferred values ​​(14%, 0.3%, 7.5) all reach higher values, proving that this combination can achieve a better balance between enzymatic hydrolysis efficiency and product activity. When the parameters are lower or higher than the range of this scheme, the thrombin inhibition rate is lower than 45%, and the physicochemical properties also decrease significantly, further verifying the rationality and necessity of the parameter range of this scheme.

[0117] Test Example 7: Comparison of antioxidant activity with other commercially available myocardial peptides

[0118] To clarify the antioxidant advantages of the horse cardiac peptide of this invention compared with similar products in the prior art, commercially available porcine cardiac peptide powder, sheep cardiac peptide powder, and bovine cardiac peptide powder were selected, and their antioxidant activity was detected using the same DPPH method and ABTS method as in test examples 1 and 2. The results are shown in Table 7 below:

[0119]

[0120] Table 7

[0121] As shown in Table 7 above, the AO values ​​of the horse myocardial peptide powder prepared by this method in the DPPH and ABTS systems are significantly lower than those of commercially available pig, bovine, and sheep myocardial peptide powders, indicating that it has superior antioxidant activity, filling the technological gap in horse myocardial peptide products, and has outstanding performance advantages compared with existing similar products.

[0122] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. A method for preparing equine cardiac peptides with antioxidant and antithrombotic effects, characterized in that, Includes the following steps: S1: Horse myocardial plasma was prepared by pretreatment of horse heart tissue; S2: Complex Enzymatic Hydrolysis and Directing Agent Regulation: A complex enzyme system and an antithrombotic directing agent are added to the horse myocardial plasma, and the enzymatic hydrolysis reaction is carried out at pH 7.3-7.7 and temperature 48-52℃. The complex enzyme system consists of alkaline protease, activated pancreas, and neutral protease. The amount of activated pancreas added is 12-16% of the total mass of the horse myocardial plasma. The antithrombotic directing agent is chitosan quaternary ammonium salt, and the amount added is 0.2-0.4% of the total mass of the horse myocardial plasma. During the enzymatic hydrolysis reaction, sodium ascorbate is added to the reaction system every 2 hours, and the amount added is 0.05% of the current total mass of the reaction system. The amount of alkaline protease added is 0.5-0.7% of the total mass of the horse myocardial plasma, and the amount of neutral protease added is 0.08-0.12% of the total mass of the horse myocardial plasma. The activated pancreas is a pancreatic enzyme preparation with an enzyme activity of 150,000 U / g, which has been activated at 4℃ for 24 hours. S3: Affinity chromatography enrichment: The enzymatic hydrolysate from step S2 is subjected to solid-liquid separation, and the resulting supernatant is purified by thrombin affinity chromatography column to collect the fraction rich in antithrombotic peptides. S4: Dry the components obtained in step S3 to obtain horse myocardial peptide powder.

2. The method for preparing equine cardiac peptides with antioxidant and antithrombotic effects according to claim 1, characterized in that: In step S2, the amount of alkaline protease added is 0.6% of the total mass of horse myocardial plasma, and the amount of neutral protease added is 0.1% of the total mass of horse myocardial plasma.

3. The method for preparing equine cardiac peptides with antioxidant and antithrombotic effects according to claim 1, characterized in that: In step S2, the amount of activated pancreas added is 14% of the total mass of horse myocardial plasma, and the amount of antithrombotic directing agent added is 0.3% of the total mass of horse myocardial plasma.

4. The method for preparing equine cardiac peptides with antioxidant and antithrombotic effects according to claim 1 or 3, characterized in that: In step S2, the pH value of the enzymatic hydrolysis reaction is 7.

5.

5. The method for preparing equine cardiac peptides with antioxidant and antithrombotic effects according to claim 1, characterized in that: In step S3, the elution process of the thrombin affinity chromatography column is performed by gradient elution using a Tris-HCl buffer containing 0.5 mol / L NaCl and with a pH of 7.

4.

6. The method for preparing equine cardiac peptides with antioxidant and antithrombotic effects according to claim 1, characterized in that: In step S1, the pretreatment includes: cutting the horse heart tissue into blocks, freezing and thawing them, and then sequentially performing ultrasonic disruption and mechanical shredding at a frequency of 40 kHz to obtain horse myocardial plasma with a particle size ≤ 500 μm.

7. The method for preparing equine cardiac peptides with antioxidant and antithrombotic effects according to claim 1, characterized in that: In step S4, the drying process specifically involves filtering and vacuum concentrating the components obtained in step S3 until the solid content is 18-22%, followed by freeze drying.

8. A horse cardiac muscle peptide with antioxidant and antithrombotic effects, characterized in that: It is a peptide mixture prepared by the method of any one of claims 1-7 for the preparation of horse cardiac peptides with antioxidant and antithrombotic effects, and simultaneously satisfies the following: half-maximal scavenging capacity (EC50) for DPPH free radicals. 50 ≤0.00267mg / L; thrombin inhibition rate ≥64.7%; peptides with a molecular weight less than 1000Da account for no less than 92% of the total peptide mass.

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