A blood source immunity active and blood pressure reducing peptide prepared by using earthworm kinase and a preparation method and application thereof

Abstract

The application discloses a kind of livestock blood source immunity active and blood pressure reducing peptide prepared by using earthworm kinase and a preparation method and application thereof.The application is prepared by pre-enzymolysis of livestock blood or livestock blood dry powder with earthworm kinase, reaction is terminated, and cooled down;after pre-enzymolysis, digestion product is obtained by simulating gastrointestinal digestion steps;centrifugation is carried out, supernatant is taken and filtered, filtrate is taken for first ultrafiltration, ultrafiltration cut-off is 5-10 ku, filtrate is taken for second ultrafiltration, ultrafiltration cut-off is 1-3 ku, filtrate is dialyzed, dialysis cut-off is 100-500 u, and freeze-drying is carried out to obtain livestock blood enzymolysis product.The application has the advantages of efficient, simple and environmental protection production method;the obtained product has high degree of hydrolysis and good immunity, antihypertensive, antioxidant and other activities, can be used as active ingredient for the production of functional food, and provides a new idea for livestock blood utilization.

Description

Technical Field

[0001] This invention belongs to the technical field of deep processing of agricultural products and comprehensive utilization of their by-products, specifically relating to a livestock and poultry blood-derived immune-active and blood pressure-lowering peptide prepared using lumbrokinase, its preparation method and application. Background Technology

[0002] Blood is one of the most significant byproducts in meat processing. my country has the world's largest pig population, with billions of pigs slaughtered annually in recent years, generating an estimated 1.8 million tons of pig blood. However, its utilization rate is extremely low, making deep processing difficult. Except for a very small portion reaching the dining table, the vast majority of pig blood is discarded, incurring high environmental costs and wasting various high-quality proteins. While hemoglobin powder has high nutritional value, its application is limited by its strong bloody taste, poor palatability, poor absorption, and low utilization rate. Enzymatic hydrolysis of pig blood protein to prepare small-molecule bioactive peptides will be a major future development direction.

[0003] Animal blood has high added value; besides being served as a dish, it is also a promising potential medicinal resource. Foreign countries started utilizing animal blood earlier, with an average utilization rate exceeding 50%. In contrast, China's processing and utilization of animal blood started later, is smaller in scale, has lower profits, fewer product varieties, and lacks innovation, thus lacking international competitiveness. However, in the context of the broader health industry, obtaining multifunctional bioactive peptides from blood has broad prospects, and market demand is expected to increase further.

[0004] Lumbrokinase has been widely used in clinical and pharmaceutical applications since its discovery, typically for its thrombolytic and anticoagulant effects. However, as a protease, it is often overlooked. Besides its thrombolytic effect, lumbrokinase can also enzymatically hydrolyze fibrin and act as a regular protease to hydrolyze proteins. Its use in preparing blood peptides has significant research value, providing new ideas for peptide production and processing, and is of great importance for the efficient utilization of blood by-products in the livestock and poultry industry.

[0005] Peptides have attracted much attention due to their diverse bioactive functions, such as immune-boosting, blood pressure-lowering, and antioxidant effects, which are among the most popular topics. Blood has numerous physiological functions, which can be used to evaluate the immune-boosting, blood pressure-lowering, and antioxidant effects of peptides, and to prepare multifunctional bioactive peptides from livestock and poultry blood.

[0006] Immunomodulatory peptides enhance the body's immune function by regulating cellular and humoral immunity. Most immunomodulatory peptides consist of 2–10 amino acid residues and exhibit good hydrophobicity. Characteristic amino acids in immunomodulatory peptides include hydrophobic amino acids, charged amino acids, and isoaromatic amino acids. Recent studies have shown that immunomodulatory protein hydrolysates or peptides can be generated from various food protein sources using endogenous enzyme autolysis. Currently, methods for evaluating the activity of immunomodulatory peptides include in vitro methods (cell models) and in vivo methods (animal models). In cell model activity evaluation, mouse macrophage RAW264.7 is the most commonly used, with TNF as an evaluation indicator. α, IL The expression levels of 6 and NO, etc.

[0007] Hypertension is a common disease in today's society. The renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) are two important systems that jointly regulate blood pressure, playing a crucial role in its control. ACE regulates blood pressure in these two different systems. ACE hydrolyzes angiotensin I, generating angiotensin II, which has the function of causing vasoconstriction and raising blood pressure. ACE inhibitory peptides inhibit the activity of ACE enzymes, thereby inhibiting the production of angiotensin II and promoting the release of kinins, ultimately achieving the effect of lowering blood pressure.

[0008] In addition to their immune-boosting and blood pressure-lowering effects, some peptides also possess antioxidant and antibacterial properties. Antioxidant peptides are widely used in preventing and scavenging free radicals, effectively reducing oxidative damage to the body. Peptides have attracted much attention due to their small molecular weight, easy absorption, strong physiological activity, multiple functions, and high safety. The preparation of blood peptides from livestock and poultry blood has enormous development potential and prospects. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a livestock and poultry blood-derived immune-active and blood pressure-lowering peptide prepared using lumbrokinase, along with its preparation method and application.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A method for preparing animal blood-derived immune-active and antihypertensive peptides using lumbrokinase includes the following steps:

[0012] (1) Pre-enzymatic hydrolysis of lumbrokinase: Mix livestock and poultry blood or dried livestock and poultry blood powder with lumbrokinase solution, stir and carry out pre-enzymatic hydrolysis. After the pre-enzymatic hydrolysis is completed, stop the reaction by boiling water bath and cool.

[0013] (2) In vitro simulated digestion:

[0014] Simulated gastric digestion stage: The cooling liquid obtained in step (1) is added to pepsin and stirred to carry out the first enzymatic hydrolysis reaction, and the pH is adjusted to terminate the simulated gastric digestion.

[0015] Simulated intestinal digestion stage: After simulating gastric digestion, pancreatic enzymes (trypsin) are added to the solution and stirred to carry out a second enzymatic reaction. Boiling is then used to terminate the simulated intestinal digestion.

[0016] (3) Ultrafiltration fractionation: After cooling the solution obtained in step (2), centrifuge, filter the supernatant, take the filtrate for the first ultrafiltration, the ultrafiltration cutoff is 5-10 kDa, take the filtrate for the second ultrafiltration, the ultrafiltration cutoff is 1-3 kDa, take the filtrate for dialysis, the dialysis cutoff is 100-500 kDa, and then freeze-dry to obtain the animal blood-derived immune-active and blood pressure-lowering peptide (animal blood enzymatic hydrolysate, denoted as blood peptide) prepared by lumbrokinase.

[0017] Preferably, in step (1), the mass-to-volume ratio of the animal blood and the lumbrokinase solution is 1:1 to 1:30 (g:mL); the mass-to-volume ratio of the dried animal blood powder and the lumbrokinase solution is 1:1 to 1:30 (g:mL); and the activity of the lumbrokinase solution used is 1000 to 200000 U / mL.

[0018] More preferably, in step (1), the mass-to-volume ratio of the animal blood and the lumbrokinase solution is 1:5 to 1:10 (g:mL); the mass-to-volume ratio of the dried animal blood powder and the lumbrokinase solution is 1:5 to 1:20 (g:mL); and the activity (concentration) of the lumbrokinase solution is 5000 to 50000 U / mL.

[0019] More preferably, in step (1), the mass-to-volume ratio of the animal blood and the lumbrokinase solution is 1:5 (g:mL); the mass-to-volume ratio of the dried animal blood powder and the lumbrokinase solution is 1:10 (g:mL); and the activity (concentration) of the lumbrokinase solution is 30000U / mL.

[0020] Preferably, in step (1), the pre-enzymatic hydrolysis temperature is 15–60°C and the time is 1–24 h;

[0021] More preferably, in step (1), the pre-enzymatic hydrolysis temperature is 37°C; and the time is 6 h.

[0022] Preferably, in step (1), the boiling water bath time is 5 to 20 minutes; more preferably, the boiling water bath time is 10 minutes.

[0023] Preferably, in step (1), the cooling time is 40 to 70 minutes. More preferably, the cooling time is 50 minutes.

[0024] Preferably, in step (2), the pH of the first enzymatic hydrolysis reaction in the simulated gastric digestion stage is 1.0 to 3.0, the time is 1 to 4 hours, and the temperature is 35 to 38°C; the pH of the second enzymatic hydrolysis reaction in the simulated intestinal digestion stage is 7.0 to 7.5, the time is 1 to 4 hours, and the temperature is 35 to 38°C.

[0025] More preferably, in step (2), the pH of the first enzymatic hydrolysis reaction in the simulated gastric digestion stage is 2.0, the time is 2 hours, and the temperature is 37°C; the pH of the second enzymatic hydrolysis reaction in the simulated intestinal digestion stage is 7.0, the time is 2 hours, and the temperature is 37°C.

[0026] Preferably, in step (2), the boiling time is 10 to 20 minutes. More preferably, the boiling time is 10 minutes.

[0027] Preferably, in step (2), the pepsin has an enzyme activity of 2500–4000 U / mg, and the pancreatin has an enzyme activity of 4–12 U / mg. More preferably, the pepsin has an enzyme activity of 3500 U / mg, and the pancreatin has an enzyme activity of 9 U / mg.

[0028] Preferably, in step (2), the amount of pepsin used is 1-10% of the mass of the cooling liquid, and the amount of pancreatin used is 1-10% of the mass of the solution after simulating gastric digestion.

[0029] More preferably, in step (2), the amount of pepsin used is 5% of the mass of the cooling liquid, and the amount of pancreatin used is 5% of the mass of the solution after simulating gastric digestion.

[0030] Preferably, in step (3), the centrifugation conditions are 4000-13000 r / min, 5-20 min, and 4-10 °C; more preferably, the centrifugation conditions are 12000 r / min, 15 min, and 4 °C.

[0031] Preferably, in step (3), the filtration conditions are filtration using a 0.22-0.45 μm filter membrane; more preferably, in step (3), the filtration conditions are filtration using a 0.45 μm filter membrane.

[0032] Preferably, in step (3), the conditions for the first ultrafiltration are a cutoff of 10 kDa and a portion of the filtrate with a molecular weight <10 kDa is taken;

[0033] Preferably, in step (3), the conditions for the second ultrafiltration are a cutoff of 3 kDa and a portion of the filtrate with a molecular weight <3 kDa is taken.

[0034] Preferably, in step (3), the dialysis conditions are a cutoff of 100u and a dialysis solution with a molecular weight >100u.

[0035] Preferably, in step (3), the temperature of the dialysis water is maintained at 4°C.

[0036] The above method yields animal blood-derived immune-active and blood pressure-lowering peptides prepared using lumbrokinase.

[0037] The above-mentioned applications of the animal blood-derived immune-active and antihypertensive peptides prepared using lumbrokinase in the preparation of antihypertensive drugs.

[0038] This invention prepares blood peptides with immune-boosting and hypotensive functional activities by pre-enzymatically hydrolyzing the blood and dried powder of various livestock and poultry under different conditions using lumbrokinase, followed by simulated gastrointestinal digestion and ultrafiltration fractionation. The process is highly efficient and simple, fully utilizing the fibrinolytic properties of lumbrokinase to increase the degree of hydrolysis of livestock and poultry blood proteins, providing new ideas and directions for the utilization of livestock and poultry blood and the development of the industry.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0040] (1) The raw materials used in this invention are animal blood and its dried powder, which have the advantages of wide availability, rich nutrition and high protein content.

[0041] (2) The present invention uses animal blood and its dry powder as raw materials, and utilizes the specificity of lumbrokinase to hydrolyze fibrin, enzymatically decomposes proteins in blood, and promotes the full release of proteins bound by fibrin.

[0042] (3) This invention innovatively utilizes lumbrokinase to pre-enzymatically hydrolyze livestock and poultry blood and its dry powder, which greatly improves the digestibility of blood proteins in the gastrointestinal tract without weakening the functional activity of blood proteins and peptides.

[0043] (4) The preparation process of the present invention is simple and convenient, low in cost, and suitable for industrial production, providing new ideas and directions for the deep processing of blood proteins of various livestock and poultry. Attached Figure Description

[0044] Figure 1 The process flow diagram for producing immune-boosting and blood pressure-lowering blood peptides derived from livestock and poultry blood provided by this invention.

[0045] Figure 2 The bar chart shows the degree of hydrolysis of the enzymatic hydrolysates of livestock and poultry blood prepared in Examples 2-6 of this invention.

[0046] Figure 3 and 4 The enzymatic hydrolysates of livestock and poultry blood prepared in Examples 2-6 of this invention have an effect on TNF in RAW264.7 cells. Bar chart showing the effect of α expression levels on survival rate.

[0047] Figure 5and Figure 6 The bar chart shows the DPPH and ABTS free radical scavenging rates of the enzymatic hydrolysates of livestock and poultry blood prepared in Examples 2-6 of this invention.

[0048] Figure 7 The in vitro hypotensive activity of the enzymatic hydrolysates of animal blood prepared in Examples 2-6 of this invention is shown in the diagram. Detailed Implementation

[0049] To better illustrate the present invention, the following description is made in conjunction with embodiments. However, it should be noted that the embodiments do not constitute a limitation on the scope of protection of the present invention. For process parameters not specifically specified, conventional techniques can be referred to.

[0050] Figure 1 The present invention provides a process flow diagram for the production of protein peptides with antihypertensive and immune-modulating biological activities from animal blood and its dried powder.

[0051] This invention provides a method for preparing animal blood-derived immune-active and blood pressure-lowering peptides using lumbrokinase, comprising the following steps:

[0052] (1) Pre-enzymatic hydrolysis of lumbrokinase: Using animal blood or its dried powder as raw material, and lumbrokinase solution as the solution, the ratio of animal blood to lumbrokinase solution (w / v) is 1:5~1:10 (g:mL); and the ratio of dried animal blood powder to lumbrokinase solution (w / v) is 1:5~1:20 (g:mL). Preheat a hot water bath to 15~60℃, and magnetically stir the reaction. The pre-enzymatic hydrolysis time is 1~24h. After the reaction is complete, stop the reaction by boiling in a water bath for 10~20min. After boiling, let it stand for 40~60min to cool to room temperature. The concentration of lumbrokinase is 1000~200000U / mL.

[0053] (2) Simulated gastrointestinal digestion: ① Simulated gastric digestion stage: Add 0.1-1.0 mol / L HCl to adjust the pH of the system to 1.5-3.0, adjust the temperature of the water bath to 35-38℃, add pepsin, stir the reaction magnetically, and the enzymatic hydrolysis time is 1-4 h; ② Simulated intestinal digestion stage: Add 0.1-1.0 mol / L NaOH to adjust the pH of the enzymatic hydrolysis system to 7.0-7.5, inhibit the activity of pepsin, terminate the simulated gastric digestion stage, add pancreatin, stir the reaction magnetically, and the enzymatic hydrolysis time is 1-4 h. After the reaction is completed, boil for 10-20 min to terminate the reaction.

[0054] (3) Ultrafiltration fractionation: After the solution is cooled to room temperature, centrifuge (8000~13000r / min, 10~20min, 4~10℃), take the supernatant and filter (0.22~0.45μm filter membrane), take the filtrate for the first ultrafiltration, the ultrafiltration cutoff is 10ku, take the filtrate with molecular weight <10ku for the second ultrafiltration, the ultrafiltration cutoff is 3ku, take the filtrate with molecular weight <3ku for dialysis desalting, the dialysis cutoff is 100~500u, freeze dry the dialysate to obtain blood peptides.

[0055] Preferably, the pre-enzymatic hydrolysis conditions in step (1) are: livestock and poultry blood: lumbrokinase solution (w / v) = 1:10 (g:mL); livestock and poultry blood powder: lumbrokinase solution (w / v) = 1:10 (g:mL), enzymatic hydrolysis temperature 37℃, boiling water bath for 10 min, and standing for 50 min. The lumbrokinase concentration is 30000U / mL.

[0056] Preferably, in step (3), the centrifugation conditions are 12000 r / min, 15 min, and 4℃.

[0057] Preferably, in step (3), the filtration condition is filtration using a 0.45 μm filter membrane.

[0058] Preferably, in step (3), the dialysis desalting cutoff is 100u, and the dialysis solution with a molecular weight >100u is used.

[0059] Preferably, in step (3), the temperature of the dialysis water should be maintained at 4°C.

[0060] Example 1

[0061] This embodiment describes a method for preparing immune-active and blood pressure-lowering peptides from livestock and poultry blood using lumbrokinase. The specific steps are as follows:

[0062] Except for the pre-enzymatic digestion, control and blank groups were designed under optimal conditions. In the blank group, the pre-enzymatic digestion solution of lumbrokinase was replaced with an equal amount of PBS (pH 7.8), and everything else was the same as the experimental group. In order to avoid the influence of lumbrokinase itself as a protein on the simulated gastrointestinal digestion products, the pre-enzymatic digestion solution of lumbrokinase in the control group was replaced with an equal amount of inactivated lumbrokinase solution, and everything else was the same as the experimental group.

[0063] (1) Pre-enzymatic hydrolysis: Animal blood (pig blood was used in this example) and its dry powder were used as raw materials and mixed at a ratio of animal blood to lumbrokinase solution (w / v) = 1:10 (g:mL); animal blood dry powder and lumbrokinase solution were mixed at a ratio of dry powder to lumbrokinase solution (w / v) = 1:10 (g:mL), and the lumbrokinase activity was 30000U / mL. The hot water bath was preheated to 37°C and the reaction was carried out with magnetic stirring for 6 hours. After the reaction was completed, the reaction was terminated by boiling in a water bath for 10 minutes. After boiling in the water bath, the mixture was allowed to stand for 50 minutes to cool to room temperature.

[0064] (2) Simulated gastrointestinal digestion: ① Simulated gastric digestion stage: Add 1.0 mol / L HCl to adjust the pH of the system to 3.0, adjust the temperature of the water bath to 37℃, add 5% pepsin (relative to the mass of the pre-digested solution), the enzyme activity of pepsin is 3500 U / mg, stir magnetically, and the enzymatic digestion time is 2 h; ② Simulated intestinal digestion stage: Add 1.0 mol / L NaOH to adjust the pH of the enzymatic digestion system to 7.0 to inhibit the enzyme activity of pepsin, terminate the simulated gastric digestion stage, add 5% trypsin (relative to the mass of the gastric digested solution), the enzyme activity of trypsin is 9 U / mg, stir magnetically, and the enzymatic digestion time is 2 h. After the reaction is completed, boil for 10 min to terminate the reaction.

[0065] (3) Ultrafiltration fractionation: After the solution is cooled to room temperature, centrifuge (13000r / min, 10min, 4℃), take the supernatant and filter (0.45μm filter membrane), take the filtrate for the first ultrafiltration, the ultrafiltration cutoff is 10ku, take the filtrate with molecular weight <10ku for the second ultrafiltration, the ultrafiltration cutoff is 3ku, take the filtrate with molecular weight <3ku for dialysis desalting, the dialysis cutoff is 100u, take the dialysate with molecular weight >100u, freeze-dry the dialysate to obtain animal blood enzymatic hydrolysate (blood peptides).

[0066] Example 1 was prepared under optimal conditions using two types of blood peptides obtained from animal blood and dried animal blood powder.

[0067] Example 1: The degree of hydrolysis of fresh livestock and poultry blood was significantly improved after pre-enzymatic hydrolysis. The degree of hydrolysis of the enzymatic hydrolysate (blood peptides) was 42.18%, and the soluble peptide content was 48.24%. The blood peptides prepared in Example 1, at a concentration of 1000 μg / mL, exhibited an ABTS·+ free radical scavenging rate of 93.21%; a DPPH· free radical scavenging rate of 53.50%; an in vitro ACE inhibition rate of 46.81%; and a peptide molecular weight of less than 1 kD comprising 62.78%. It also showed increased activity against RAW264.7 macrophage cytokine TNF. The expression level of α was 5.86 ng / mL. The blank group of this immunoassay was a culture medium without sample (89% (v / v) DMEM + 10% (v / v) FBS + 1% (v / v) penicillin and streptomycin). The survival rate of this blood peptide against RAW264.7 macrophages was 141.42%.

[0068] Example 1: After pre-enzymatic hydrolysis of livestock and poultry blood powder, the degree of hydrolysis was significantly improved. The degree of hydrolysis of the enzymatic hydrolysate (blood peptides) was 35.87%, and the soluble peptide content was 45.87%. The blood peptides prepared in Example 1, at a concentration of 1000 μg / mL, exhibited an ABTS·+ free radical scavenging rate of 91.22%; a DPPH· free radical scavenging rate of 51.14%; an in vitro ACE inhibition rate of 48.87%; and a peptide molecular weight below 1 kD comprising 60.46%. They also showed increased activity against RAW264.7 macrophage cytokine TNF. The expression level of α was 5.48 ng / mL. The blank group of this immunoassay was a culture medium without sample (89% (v / v) DMEM + 10% (v / v) FBS + 1% (v / v) penicillin and streptomycin). The survival rate of this blood peptide against RAW264.7 macrophages was 131.22%.

[0069] Blood peptides obtained from livestock and poultry blood and its dried powder after pre-enzymatic hydrolysis with lumbrokinase exhibited improvements in degree of hydrolysis, soluble peptide content, free radical scavenging rate, cell viability, in vitro ACE inhibition rate, and TNF content. The expression level of α was significantly higher than that of the control group and the blank group, indicating that the pre-enzymatic hydrolysis of lumbrokinase plays an important role in the preparation of blood peptides.

[0070]

[0071]

[0072] Example 2

[0073] This embodiment describes a method for preparing immune-active and blood pressure-lowering peptides from livestock and poultry blood using lumbrokinase. The specific steps are as follows:

[0074] (1) Pre-enzymatic hydrolysis: Livestock and poultry blood powder (pig blood was used in this example) was used as raw material. The livestock and poultry blood powder and lumbrokinase solution were mixed at a ratio of powder to solution (w / v) of 1:10 (g:mL). The lumbrokinase activity was 30,000 U / mL. The water bath was preheated to 35°C and the reaction was carried out with magnetic stirring for 6 hours. After the reaction was completed, the water bath was boiled for 10 minutes to terminate the reaction. After the water bath, the water bath was allowed to stand for 50 minutes to cool to room temperature.

[0075] (2) Simulated gastrointestinal digestion: ① Simulated gastric digestion stage: Add 1.0 mol / L HCl to adjust the pH of the system to 3.0, adjust the temperature of the water bath to 37℃, add 5% pepsin (relative to the mass of the pre-digested solution), the enzyme activity of pepsin is 3500 U / mg, stir magnetically, and the enzymatic digestion time is 2 h; ② Simulated intestinal digestion stage: Add 1.0 mol / L NaOH to adjust the pH of the enzymatic digestion system to 7.0 to inhibit the enzyme activity of pepsin, terminate the simulated gastric digestion stage, add 5% trypsin (relative to the mass of the gastric digested solution), the enzyme activity of trypsin is 9 U / mg, stir magnetically, and the enzymatic digestion time is 2 h. After the reaction is completed, boil for 10 min to terminate the reaction.

[0076] (3) Ultrafiltration fractionation: After the solution is cooled to room temperature, centrifuge (13000r / min, 10min, 4℃), take the supernatant and filter (0.45μm filter membrane), take the filtrate for the first ultrafiltration, the ultrafiltration cutoff is 10ku, take the filtrate with molecular weight <10ku for the second ultrafiltration, the ultrafiltration cutoff is 3ku, take the filtrate with molecular weight <3ku for dialysis desalting, the dialysis cutoff is 100u, take the dialysate with molecular weight >100u, freeze-dry the dialysate to obtain animal blood enzymatic hydrolysate (blood peptides).

[0077] In Example 2, the degree of hydrolysis of the animal blood enzymatic hydrolysate (blood peptides) obtained in step 3 was 35.42%, and the soluble peptide content was 43.92%. The animal blood enzymatic hydrolysate prepared in Example 2, at a concentration of 1000 μg / mL, showed increased levels of the macrophage RAW264.7 cytokine TNF. The expression level of α was 5.38 ng / mL, which was 439.67% higher than that of the blank group in the immunoassay experiment. The blank group in the immunoassay experiment consisted of culture medium without samples (89% (v / v) DMEM + 10% (v / v) FBS + 1% (v / v) penicillin and streptomycin). Figure 3 As shown in the figure. The survival rate of this hemopeptide against RAW264.7 macrophages was 131.22%, as... Figure 4 As shown in the figure.

[0078] Example 3

[0079] This embodiment describes a method for preparing immune-active and blood pressure-lowering peptides from livestock and poultry blood using lumbrokinase. The specific steps are as follows:

[0080] (1) Pre-enzymatic hydrolysis: Take dried animal blood (pig blood in this example) as raw material, mix dried animal blood powder with lumbrokinase solution at a ratio of dried powder:lumbrokinase solution (w / v) = 1:10 (g:mL), the lumbrokinase activity is 30000U / mL; preheat the hot water bath to 40℃, stir the reaction with magnetic force, the pre-enzymatic hydrolysis time is 6h, after the reaction is completed, boil in water bath for 10min to terminate the reaction, and let stand for 50min after boiling in water bath to cool to room temperature.

[0081] (2) Simulated gastrointestinal digestion: ① Simulated gastric digestion stage: Add 1.0 mol / L HCl to adjust the pH of the system to 3.0, adjust the temperature of the water bath to 37℃, add 5% pepsin (relative to the mass of the pre-digested solution), the enzyme activity of pepsin is 3500 U / mg, stir magnetically, and the enzymatic digestion time is 2 h; ② Simulated intestinal digestion stage: Add 1.0 mol / L NaOH to adjust the pH of the enzymatic digestion system to 7.0 to inhibit the enzyme activity of pepsin, terminate the simulated gastric digestion stage, add 5% trypsin (relative to the mass of the gastric digested solution), the enzyme activity of trypsin is 9 U / mg, stir magnetically, and the enzymatic digestion time is 2 h. After the reaction is completed, boil for 10 min to terminate the reaction.

[0082] (3) Ultrafiltration fractionation: After the solution is cooled to room temperature, centrifuge (13000r / min, 10min, 4℃), take the supernatant and filter (0.45μm filter membrane), take the filtrate for the first ultrafiltration, the ultrafiltration cutoff is 10ku, take the filtrate with molecular weight <10ku for the second ultrafiltration, the ultrafiltration cutoff is 3ku, take the filtrate with molecular weight <3ku for dialysis desalting, the dialysis cutoff is 100u, take the dialysate with molecular weight >100u, freeze dry the dialysate to obtain animal blood enzymatic hydrolysate (blood peptides).

[0083] In Example 3, the degree of hydrolysis of the livestock and poultry blood enzymatic hydrolysate (blood peptides) obtained in step 3 was 32.71%, and the soluble peptide content was 42.74%. At a concentration of 1000 μg / mL, the livestock and poultry blood enzymatic hydrolysate prepared in Example 3 showed increased levels of the macrophage RAW264.7 cytokine TNF. The expression level of α was 6.57 ng / mL, which was 536.92% higher than that of the blank group in the immunization experiment. The blank group in the immunization experiment consisted of culture medium without samples (89% (v / v) DMEM + 10% (v / v) FBS + 1% (v / v), with streptomycin and penicillin as the penicillin and antimicrobial antibodies). Figure 3 As shown in the figure. The survival rate of this hemopeptide against RAW264.7 macrophages was 101.49%, as... Figure 4 As shown in the figure.

[0084] Example 4

[0085] This embodiment describes a method for preparing immune-active and blood pressure-lowering peptides from livestock and poultry blood using lumbrokinase. The specific steps are as follows:

[0086] (1) Pre-enzymatic hydrolysis: Take dried animal blood (pig blood in this example) as raw material, mix dried animal blood powder with lumbrokinase solution at a ratio of dried powder:lumbrokinase solution (w / v) = 1:10 (g:mL), the lumbrokinase activity is 30000U / mL; preheat the hot water bath to 25℃, stir the reaction with magnetic force, the pre-enzymatic hydrolysis time is 8h, after the reaction is completed, boil water bath for 10min to terminate the reaction, and let stand for 50min after boiling water bath to cool to room temperature.

[0087] (2) Simulated gastrointestinal digestion: ① Simulated gastric digestion stage: Add 1.0 mol / L HCl to adjust the pH of the system to 3.0, adjust the temperature of the water bath to 37℃, add 5% pepsin (relative to the mass of the pre-digested solution), the enzyme activity of pepsin is 3500 U / mg, stir magnetically, and the enzymatic digestion time is 2 h; ② Simulated intestinal digestion stage: Add 1.0 mol / L NaOH to adjust the pH of the enzymatic digestion system to 7.0 to inhibit the enzyme activity of pepsin, terminate the simulated gastric digestion stage, add 5% trypsin (relative to the mass of the gastric digested solution), the enzyme activity of trypsin is 9 U / mg, stir magnetically, and the enzymatic digestion time is 2 h. After the reaction is completed, boil for 10 min to terminate the reaction.

[0088] (3) Ultrafiltration fractionation: After the solution is cooled to room temperature, centrifuge (13000r / min, 15min, 4℃), take the supernatant and filter (0.45μm filter membrane), take the filtrate for the first ultrafiltration, the ultrafiltration cutoff is 10ku, take the filtrate with molecular weight <10ku for the second ultrafiltration, the ultrafiltration cutoff is 3ku, take the filtrate with molecular weight <3ku for dialysis desalting, the dialysis cutoff is 100u, take the dialysate with molecular weight >100u, freeze-dry the dialysate to obtain animal blood enzymatic hydrolysate (blood peptides).

[0089] In Example 4, the degree of hydrolysis of the livestock and poultry blood enzymatic hydrolysate (blood peptides) obtained in step 3 was 28.42%, and the soluble peptide content of the pre-hydrolysate was 38.43%. The livestock and poultry blood enzymatic hydrolysate prepared in Example 4, at a concentration of 1000 μg / mL, showed increased levels of the macrophage RAW264.7 cytokine TNF. The expression level of α was 3.90 ng / mL, which was 318.72% higher than that of the blank group in the immunoassay experiment. The blank group in the immunoassay experiment consisted of culture medium without samples (89% (v / v) DMEM + 10% (v / v) FBS + 1% (v / v) penicillin and streptomycin). Figure 3 As shown in the figure. This blood peptide showed a survival rate of 104.47% for RAW264.7 macrophages, as... Figure 4 As shown in the figure.

[0090] Example 5

[0091] This embodiment describes a method for preparing immune-active and blood pressure-lowering peptides from livestock and poultry blood using lumbrokinase. The specific steps are as follows:

[0092] (1) Take dried animal blood (pig blood is used in this example) as raw material. Mix dried animal blood powder with lumbrokinase solution at a ratio of dried powder:lumbrokinase solution (w / v) = 1:10 (g:mL). The lumbrokinase activity is 30000U / mL. Preheat the hot water bath to 30°C, stir the reaction with magnetic force, and pre-enzymatic hydrolysis time is 6h. After the reaction is completed, boil in water for 10min to terminate the reaction. After boiling in water bath, let stand for 50min to cool to room temperature.

[0093] (2) Simulated gastrointestinal digestion: ① Simulated gastric digestion stage: Add 1.0 mol / L HCl to adjust the pH of the system to 3.0, adjust the temperature of the water bath to 37℃, add 5% pepsin (relative to the mass of the pre-digested solution), the enzyme activity of pepsin is 3500 U / mg, stir magnetically, and the enzymatic digestion time is 2 h; ② Simulated intestinal digestion stage: Add 1.0 mol / L NaOH to adjust the pH of the enzymatic digestion system to 7.0 to inhibit the enzyme activity of pepsin, terminate the simulated gastric digestion stage, add 5% trypsin (relative to the mass of the gastric digested solution), the enzyme activity of trypsin is 9 U / mg, stir magnetically, and the enzymatic digestion time is 2 h. After the reaction is completed, boil for 10 min to terminate the reaction.

[0094] (3) Ultrafiltration fractionation: After the solution is cooled to room temperature, centrifuge (13000r / min, 15min, 4℃), take the supernatant and filter (0.45μm filter membrane), take the filtrate for the first ultrafiltration, the ultrafiltration cutoff is 10ku, take the filtrate with molecular weight <10ku for the second ultrafiltration, the ultrafiltration cutoff is 3ku, take the filtrate with molecular weight <3ku for dialysis desalting, the dialysis cutoff is 100u, take the dialysate with molecular weight >100u, freeze-dry the dialysate to obtain animal blood enzymatic hydrolysate (blood peptides).

[0095] In Example 5, the degree of hydrolysis of the animal blood enzymatic hydrolysate (blood peptides) obtained in step 3 was 29.10%, and the soluble peptide content was 36.28%. The animal blood enzymatic hydrolysate prepared in Example 5, at a concentration of 1000 μg / mL, showed increased levels of the macrophage RAW264.7 cytokine TNF. The expression level of α was 5.44 ng / mL, which was 444.57% higher than that of the blank group in the immunoassay experiment. The blank group in the immunoassay experiment consisted of culture medium without samples (89% (v / v) DMEM + 10% (v / v) FBS + 1% (v / v) penicillin and streptomycin). Figure 3 As shown in the figure. This blood peptide showed a 92.81% survival rate in RAW264.7 macrophages, as... Figure 4 As shown in the figure.

[0096] Example 6

[0097] This embodiment describes a method for preparing immune-active and blood pressure-lowering peptides from livestock and poultry blood using lumbrokinase. The specific steps are as follows:

[0098] (1) Take dried animal blood (pig blood is used in this example) as raw material. Mix dried animal blood powder with lumbrokinase solution at a ratio of dried powder:lumbrokinase solution (w / v) = 1:10 (g:mL). The lumbrokinase activity is 30000U / mL. Preheat the hot water bath to 45°C, stir the reaction with magnetic force, and pre-enzymatic hydrolysis time is 6h. After the reaction is completed, boil in water for 10min to terminate the reaction. After boiling in water bath, let stand for 50min to cool to room temperature.

[0099] (2) Simulated gastrointestinal digestion: ① Simulated gastric digestion stage: Add 1.0 mol / L HCl to adjust the pH of the system to 3.0, adjust the temperature of the water bath to 37℃, add 5% pepsin (relative to the mass of the pre-digested solution), the enzyme activity of pepsin is 3500 U / mg, stir magnetically, and the enzymatic digestion time is 2 h; ② Simulated intestinal digestion stage: Add 1.0 mol / L NaOH to adjust the pH of the enzymatic digestion system to 7.0 to inhibit the enzyme activity of pepsin, terminate the simulated gastric digestion stage, add 5% trypsin (relative to the mass of the gastric digested solution), the enzyme activity of trypsin is 9 U / mg, stir magnetically, and the enzymatic digestion time is 2 h. After the reaction is completed, boil for 10 min to terminate the reaction.

[0100] (3) Ultrafiltration fractionation: After the solution is cooled to room temperature, centrifuge (13000r / min, 15min, 4℃), take the supernatant and filter (0.45μm filter membrane), take the filtrate for the first ultrafiltration, the ultrafiltration cutoff is 10ku, take the filtrate with molecular weight <10ku for the second ultrafiltration, the ultrafiltration cutoff is 3ku, take the filtrate with molecular weight <3ku for dialysis desalting, the dialysis cutoff is 100u, take the dialysate with molecular weight >100u, freeze-dry the dialysate to obtain animal blood enzymatic hydrolysate (blood peptides).

[0101] In Example 6, the degree of hydrolysis of the livestock and poultry blood enzymatic hydrolysate (blood peptides) obtained in step 3 was 29.16%, and the soluble peptide content was 37.86%. At a concentration of 1000 μg / mL, the livestock and poultry blood enzymatic hydrolysate prepared in Example 6 showed increased levels of the macrophage RAW264.7 cytokine TNF. The expression level of α was 4.76 ng / mL, which was 389.00% higher than that of the blank group in the immunoassay experiment. The blank group in the immunoassay experiment consisted of culture medium without samples (89% (v / v) DMEM + 10% (v / v) FBS + 1% (v / v) penicillin and streptomycin). Figure 3As shown in the figure. The survival rate of this hemopeptide against RAW264.7 macrophages was 102.92%, as... Figure 4 As shown in the figure.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing immunomodulatory and antihypertensive peptides from livestock and poultry blood using lumbrokinase, characterized in that, Includes the following steps: (1) Pre-enzymatic hydrolysis of lumbrokinase: Livestock and poultry blood or dried livestock and poultry blood powder is mixed with lumbrokinase solution and stirred for pre-enzymatic hydrolysis. After pre-enzymatic hydrolysis, the reaction is terminated by boiling water bath and then cooled. The mass-volume ratio of the livestock and poultry blood to the lumbrokinase solution is 1:1 to 1:30 g:mL. The mass-volume ratio of the dried livestock and poultry blood powder to the lumbrokinase solution is 1:1 to 1:30 g:mL. The activity of the lumbrokinase solution used is 1000~200000U / mL. The temperature of the pre-enzymatic hydrolysis is 15~60℃ and the time is 1~24h. The boiling water bath time is 5~20min. The cooling time is 40~70min. The selected livestock and poultry blood and dried livestock and poultry blood powder are from pig blood. (2) In vitro simulated digestion: Simulated gastric digestion stage: The cooling liquid obtained in step (1) is added to pepsin and stirred to carry out the first enzymatic hydrolysis reaction, and the pH is adjusted to terminate the simulated gastric digestion; the pH of the first enzymatic hydrolysis reaction in the simulated gastric digestion stage is 1.0 to 3.0, the time is 1 to 4 hours, and the temperature is 35 to 38°C. Simulated intestinal digestion stage: After simulating gastric digestion, pancreatic enzymes are added to the solution and stirred to carry out a second enzymatic hydrolysis reaction. The simulated intestinal digestion is terminated by boiling. The pH of the second enzymatic hydrolysis reaction in the simulated intestinal digestion stage is 7.0-7.5, the time is 1-4 hours, and the temperature is 35-38℃. The pepsin has an enzyme activity of 2500–4000 U / mg, and the pancreatin has an enzyme activity of 4–12 U / mg; the amount of pepsin used is 1–10% of the mass of the cooling liquid, and the amount of pancreatin used is 1–10% of the mass of the solution after simulated gastric digestion; (3) Ultrafiltration fractionation: After cooling the solution obtained in step (2), centrifuge, filter the supernatant, and perform a first ultrafiltration on the filtrate with an ultrafiltration cutoff of 10 kDa. Perform a second ultrafiltration on the filtrate with a molecular weight <10 kDa with an ultrafiltration cutoff of 3 kDa. Perform dialysis on the filtrate with a molecular weight <3 kDa with a dialysis cutoff of 100 kDa. Perform freeze-drying on the dialysis solution with a molecular weight >100 kDa. Obtain the animal blood-derived immune-active and blood pressure-lowering peptides prepared using lumbrokinase. The centrifugation conditions are 4000–13000 r / min, 5–20 min, and 4–10 °C. The filtration conditions are filtration through a 0.22–0.45 μm filter membrane.

2. The method for preparing animal blood-derived immune-active and antihypertensive peptides using lumbrokinase according to claim 1, characterized in that, In step (2), the boiling time is 10 to 20 minutes.

3. The animal blood-derived immune-active and blood pressure-lowering peptide prepared by the method according to any one of claims 1 to 2.

4. The application of the animal blood-derived immune-active and antihypertensive peptides prepared using lumbrokinase as described in claim 3 in the preparation of antihypertensive drugs.

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