Deer myocardial peptide, preparation method and application thereof

Abstract

The application relates to the field of small-molecule peptides, and discloses a deer heart muscle peptide as well as a preparation method and application thereof. The preparation method of the deer heart muscle peptide comprises the following steps: after frozen deer hearts are thawed, the fascia, heart tube and fat are removed, the meat is cut into pieces, washed and then minced into meat paste; the meat paste is subjected to enzymolysis, enzyme inactivation, centrifugation, supernatant taking, and drying to obtain the deer heart muscle peptide; the protease used for the enzymolysis is papain and flavor enzyme, the mass ratio of the material to the liquid is 1:3-3.5, and the enzymolysis time is 4-5 h; the papain and the flavor enzyme are used for the enzymolysis in a ratio of 4-4.5 ‰:2-2.5 ‰ to obtain the deer heart muscle peptide product with good flavor; the deer heart muscle peptide is coated with a composite wall material, combined with ultrasonic treatment and drying technology, and finally the deer heart muscle peptide with high antioxidant capacity, good antibacterial performance, high storage stability, low cost and the like is prepared, which can be applied to the microcapsule process in large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of small molecule peptide technology, specifically relating to a deer cardiac muscle peptide, its preparation method, and its application. Background Technology

[0002] Deer cardiac muscle peptide is a small molecule peptide product from deer heart. For peptide products, bitterness and astringency have a significant impact and are the main factors affecting their flavor. In addition, peptide products are easily deactivated by light, heat, oxygen, acid and alkaline environments, or enzymes during processing and storage. Microencapsulation technology encapsulates deer cardiac muscle peptide in tiny capsules formed by wall materials, creating a physical barrier to prevent direct contact with the external environment, thereby improving its stability and sustained-release properties during processing and storage. However, traditional encapsulation methods, including spray drying, emulsification, and composite coagulation, have problems such as low encapsulation rate, high energy consumption, and complex processes, making them unsuitable for industrial production. Furthermore, most existing microcapsule wall materials use soy protein, whey protein, or casein, but these wall materials themselves have weak antioxidant properties or lack oxygen barrier capabilities. As a result, the antioxidant effect of the prepared deer cardiac muscle peptide microcapsules is not ideal during storage, affecting the application of the product. Summary of the Invention

[0003] To address the shortcomings mentioned in the background art, the present invention aims to provide a deer myocardial peptide, its preparation method, and its application. The deer myocardial peptide product with good flavor is obtained by enzymatic hydrolysis using papain and flavor enzymes added in a ratio of 4~4.5‰:2~2.5‰. Furthermore, a composite wall material is coated onto the surface of the deer myocardial peptide, and combined with ultrasonic treatment and drying technology, a deer myocardial peptide with high antioxidant capacity, good antibacterial properties, high storage stability, and low cost is prepared, which can be applied in large-scale production.

[0004] The objective of this invention can be achieved through the following technical solutions: A method for preparing deer cardiac muscle peptide includes the following steps: (1) Take frozen deer heart, thaw it, remove the fascia, heart tube and fat, cut it into pieces, wash it and mince it into meat paste; (2) The minced meat was enzymatically hydrolyzed, enzymes were inactivated, centrifuged, the supernatant was collected and dried to prepare deer myocardial peptide; The proteases used in the enzymatic hydrolysis are papain and flavor enzyme, with a material-to-liquid mass ratio of 1:3 to 3.5 and a hydrolysis time of 4 to 5 hours.

[0005] Preferably, the ratio of papain to flavor enzyme is 4~4.5‰: 2~2.5‰.

[0006] Preferably, the protein content after enzymatic hydrolysis is 3.83~3.94%, the protein recovery rate is 90.23~93.36%, the molecular weight distribution of 202~3000 Da is 90.6~93.8%, and the average molecular weight is about 500 Da.

[0007] Preferably, the drying method is one of spray drying, freeze drying, or low-temperature vacuum belt drying.

[0008] A deer cardiac muscle peptide, prepared by the method described above, wherein the peptide sequence of the deer cardiac muscle peptide includes DWRPPRQ, WHEP, RHPYF and NGPDHWHE, the amino acid sequence of DWRPPRQ is shown in SEQ ID NO 1, the amino acid sequence of WHEP is shown in SEQ ID NO 2, the amino acid sequence of RHPYF is shown in SEQ ID NO 3, and the amino acid sequence of NGPDHWHE is shown in SEQ ID NO 4.

[0009] An application of deer myocardial peptide as described above involves mixing the deer myocardial peptide with a composite wall material and then subjecting the mixture to ultrasonic treatment. The composite wall material is then coated onto the surface of the deer myocardial peptide and dried to form deer myocardial peptide microcapsules. The ratio of the core material to the composite wall material of the deer myocardial peptide microcapsule is 1:3~5; the ultrasonic treatment time is 30~60min, the power is 125~175W, and the temperature is 40~60℃.

[0010] Preferably, the composite wall material is a resistant dextrin and a chitosan derivative.

[0011] Preferably, the composite wall material is resistant dextrin, modified zein, and chitosan derivative.

[0012] Preferably, the method for preparing the modified zein includes the following steps: (1) Corn gluten powder was treated with α-amylase and acetone to remove starch and decolorize. It was then extracted with 70% ethanol solution and centrifuged at 3950~4100 r / min for 20~30 min. The supernatant was collected and then extracted with ice water. The precipitate was freeze-dried to prepare corn gliadin. (2) Dissolve zein in 70% ethanol solution and then slowly add it to 5% glucose solution and stir to react. Place it in a water bath at 55-65℃ and sonicate for 5-8 minutes to prepare modified zein. The mass ratio of glucose to zein is 1:8~12; the stirring reaction time is 20~40 min; and the ultrasonic power is 400~500W.

[0013] Preferably, the preparation method of the chitosan derivative includes the following steps: dissolving chitosan in an aqueous acetic acid solution, stirring magnetically until a clear and transparent solution is obtained, then adding ascorbic acid and hydrogen peroxide, and stirring continuously for 25-40 minutes under nitrogen protection, followed by adding rosmarinic acid and continuing the reaction at room temperature for 10-12 hours, after the reaction is completed, introducing the obtained polymer solution into a dialysis bag and immersing it in a glass container containing deionized water for dialysis, and finally freeze-drying to obtain the chitosan derivative; The molar ratio of chitosan repeating units to rosmarinic acid is 1:1; the concentration of ascorbic acid is 0.2~0.4 mmol / L; and the concentration of hydrogen peroxide is 600~1000 mmol / L.

[0014] The beneficial effects of this invention are: The deer cardiac peptide prepared in this invention has a significant protective effect against H2O2-induced oxidative stress damage in H9C2 cardiomyocytes. The mechanism is as follows: by reducing the accumulation of intracellular reactive oxygen species (ROS) and the level of lipid peroxidation products (MDA), it enhances the activity of antioxidant enzymes (SOD) to maintain the oxidation-antioxidant balance; simultaneously, it reduces the release of cardiac enzymes (LDH, CK, AST) to protect cell membrane integrity and inhibit apoptosis and necrosis.

[0015] The deer cardiac muscle peptide prepared in this invention caused no damage to RAW264.7 cells and could promote cell proliferation to a certain extent. Regarding oxidative damage, the deer cardiac muscle peptide had no significant effect on MDA content, indicating that it had no significant oxidative damage effect on RAW264.7 cells. In terms of immune function indicators, NO secretion increased with increasing small molecule peptide concentration, exhibiting a concentration-dependent effect mimicking the LPS immune activation effect; the phagocytic index was significantly increased, enhancing macrophage immune function; the secretion of TNF-α, IL-1β, and IL-6 increased with increasing concentration, indicating that the deer cardiac muscle peptide could induce cytokine production and exert an immunostimulatory effect. Simultaneously, the deer cardiac muscle peptide could promote ROS production, enhance cell adhesion, and promote macrophage proliferation by increasing the proportion of cells in S phase. In summary, the deer cardiac muscle peptide can activate RAW264.7 macrophages and exert immunomodulatory effects through multiple pathways, including proliferation, immune factor secretion, phagocytosis, and adhesion, making it a potential immune enhancer.

[0016] The deer cardiac peptide prepared in this invention showed significant protective effects against myocardial injury in mice. In the model group, the infarct area was the most severe. Compared to the model group, the infarct area was reduced in all dose groups of deer cardiac peptide, demonstrating a clear protective effect against myocardial injury. Myocardial enzyme detection results showed that deer cardiac peptide could reduce serum CK, AST, and LDH levels, thus improving myocardial injury in mice. Regarding oxidative damage, treatment with different concentrations of deer cardiac peptide significantly reduced serum MDA levels and significantly increased SOD levels compared to the model group, indicating that deer cardiac peptide improves acute myocardial ischemia through its antioxidant effects. Cytokine detection showed decreased expression levels of TNF-α, IL-6, and IL-1β in all dose groups of deer cardiac peptide, suggesting that deer cardiac peptide can improve acute myocardial ischemia through its anti-inflammatory effects. In conclusion, deer cardiac peptide can improve isoproterenol-induced myocardial injury in mice from multiple dimensions, including myocardial infarction area, myocardial enzymes, free radical scavenging, and cytokine regulation.

[0017] This invention utilizes papain and flavor enzymes added in a ratio of 4~4.5‰:2~2.5‰ for enzymatic hydrolysis, resulting in lower bitterness and astringency levels and better flavor. This invention utilizes glucose as a carbonyl donor in a glycosylation reaction to modify zein through a wet glycosylation process. Zein is widely found as a byproduct of corn starch processing and is a natural plant protein with advantages such as easy availability, high cost-effectiveness, and safety. Its protein contains over 50% hydrophobic amino acid residues, and its unique amino acid composition can serve as a natural plant antioxidant. Glucose is a widely available reducing monosaccharide. After modification with glucose, the number of hydrogen bonds, disulfide bonds, and hydrophobic bonds between zein molecules increases, the molecular forces between zein and glucose are enhanced, the spatial structure becomes more compact, and the permeability is reduced, further improving the antioxidant properties of zein. This invention also utilizes a free radical-induced grafting method to covalently graft chitosan and rosmarinic acid in an ascorbic acid / hydrogen peroxide system to prepare chitosan derivatives. Chitosan is a deacetylated product of chitin and possesses unique antibacterial and bioactive properties. Rosmarinic acid, a natural antioxidant with biocompatibility and biodegradability, possesses antioxidant, anti-inflammatory, and antibacterial biological activities. Grafting reactions can effectively reduce hydrogen bonds within and between chitosan molecules, thereby improving chitosan's solubility and synergistically enhancing its excellent antioxidant and antibacterial properties. This invention utilizes resistant dextrin, modified zein, and chitosan derivatives as wall materials. Resistant dextrin, a low-calorie dextran, can be used as a dietary fiber additive, promoting intestinal health and slowing down carbohydrate absorption. Furthermore, resistant dextrin exhibits low viscosity, high water solubility, and thermal stability. Encapsulating deer cardiac peptides with a composite wall material of resistant dextrin, modified zein, and chitosan derivatives improves the encapsulation rate. Combined with ultrasonic treatment and drying techniques, microcapsules containing deer cardiac peptides with high antioxidant capacity, good antibacterial properties, high storage stability, and low cost are prepared, making them suitable for large-scale production. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a graph showing the relationship between deer myocardial peptide and H2O2 concentration in this invention; Figure 2 This is a graph showing the relationship between the concentration of deer cardiac peptide and myocardial cell viability in this invention; Figure 3This is a graph showing the relationship between deer cardiac peptide pretreatment and H2O2-induced cardiomyocyte viability according to the present invention. Figure 4 This is a graph showing the relationship between deer cardiac muscle peptide pretreatment and intracellular MDA and SOD content in this invention. Figure 5 This invention presents a graph showing the relationship between deer cardiac muscle peptide pretreatment and intracellular AST, CK, and LDH levels. In graph A, deer cardiac muscle peptide pretreatment is shown as the relationship between intracellular AST levels; in graph B, deer cardiac muscle peptide pretreatment is shown as the relationship between intracellular CK levels; and in graph C, deer cardiac muscle peptide pretreatment is shown as the relationship between intracellular LDH levels. Figure 6 This is a diagram showing the relationship between deer cardiac peptide pretreatment and H2O2-induced apoptosis and necrosis of cardiomyocytes according to the present invention. Figure 7 This is a graph showing the relationship between deer cardiac peptide pretreatment and H2O2-induced intracellular reactive oxygen species content in cardiomyocytes according to the present invention. Figure 8 This is a diagram showing the proliferation relationship of RAW264.7 cells after treatment with deer cardiac muscle peptide according to the present invention; Figure 9 This is a graph showing the relationship between MDA content in RAW264.7 cells after treatment with deer cardiac muscle peptide according to the present invention. Figure 10 This is a graph showing the relationship between NO secretion in RAW264.7 cells after treatment with deer cardiac muscle peptide according to the present invention; Figure 11 This is a graph showing the relationship between TNF-α, IL-1β, and IL-6 secretion levels in RAW264.7 cells after treatment with deer cardiac peptide according to the present invention. In the graph, A is the relationship between IL-6 secretion level, B is the relationship between IL-1β secretion level, and C is the relationship between TNF-α secretion level. Figure 12 This is a diagram showing the relationship between the phagocytic function of RAW264.7 cells after treatment with deer myocardial peptide according to the present invention. Figure 13 This is a graph showing the relationship between ROS content in RAW264.7 cells after treatment with deer myocardial peptide according to the present invention. Figure 14 This is a graph showing the relationship between the adhesion ability of RAW264.7 cells after treatment with deer cardiac peptide according to the present invention. Figure 15 This is a peak pattern diagram of G0 / 1, S, and G2 / M in RAW264.7 cells after treatment with deer cardiac muscle peptide according to the present invention; Figure 16 This is a cell cycle distribution diagram of RAW264.7 cells after treatment with deer cardiac peptide according to the present invention. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] In the embodiments and comparative examples of this application, the flavor enzyme is a flavor protease purchased from Nanning Pangbo Biotechnology Co., Ltd.

[0022] Example 1: A method for preparing deer cardiac muscle peptide includes the following steps: (1) Take frozen deer heart, thaw it, remove the fascia, heart tube and fat, cut it into pieces, wash it and mince it into meat paste; (2) The minced meat was enzymatically hydrolyzed, enzyme-inactivated, centrifuged, the supernatant was collected and dried to prepare deer myocardial peptide. The enzymatic hydrolysis conditions were as follows: the ratio of papain to flavor enzyme was 4‰:2‰, the material-to-liquid mass ratio was 1:3, the hydrolysis time was 4h, and the protein content was 3.92%, the protein recovery rate was 92.40%, the peptide yield was 86.67%, and the molecular weight distribution of 202~3000Da was 93.8%.

[0023] Example 2: A method for preparing deer cardiac muscle peptide includes the following steps: (1) Take frozen deer heart, thaw it, remove the fascia, heart tube and fat, cut it into pieces, wash it and mince it into meat paste; (2) The minced meat was enzymatically hydrolyzed, enzyme-inactivated, centrifuged, the supernatant was collected and dried to prepare deer myocardial peptide. The enzymatic hydrolysis conditions were: the ratio of papain to flavor enzyme was 4‰:2‰, the material-to-liquid mass ratio was 1:3, the hydrolysis time was 5h, and the protein content was 3.83%, the protein recovery rate was 90.23%, and the molecular weight distribution of 202~3000Da was 90.6%.

[0024] Example 3 A method for preparing deer cardiac muscle peptide includes the following steps: (1) Take frozen deer heart, thaw it, remove the fascia, heart tube and fat, cut it into pieces, wash it and mince it into meat paste; (2) The minced meat was enzymatically hydrolyzed, enzyme-inactivated, centrifuged, the supernatant was collected and dried to prepare deer myocardial peptide. The enzymatic hydrolysis conditions were as follows: the ratio of papain to flavor enzyme was 4.5‰:2.5‰, the material-to-liquid mass ratio was 1:3, the hydrolysis time was 4h, and the protein content was 3.94%, the protein recovery rate was 93.36%, and the molecular weight distribution of 202~3000Da was 93.0%.

[0025] Example 4 A method for preparing modified zein includes the following steps: (1) Corn gluten powder was treated with α-amylase and acetone to remove starch and decolorize. It was then extracted with 70% ethanol solution and centrifuged at 4000 r / min for 30 min. The supernatant was collected and then extracted with ice water. The precipitate was freeze-dried to prepare corn gliadin. (2) Dissolve zein in 70% ethanol solution. Add zein slowly to 5% glucose solution at a mass ratio of 1:10 and stir for 30 min. Place in a 60℃ water bath and sonicate at 450W for 5 min to prepare modified zein.

[0026] Example 5 A method for preparing a chitosan derivative includes the following steps: 1 g of chitosan was dissolved in an aqueous acetic acid solution (1% w / v) and magnetically stirred until a clear and transparent solution was obtained. Then, 0.3 mmol / L ascorbic acid and 800 mmol / L hydrogen peroxide were added and stirred continuously for 30 min under nitrogen protection. Subsequently, rosmarinic acid was added at a molar ratio of chitosan repeating unit to rosmarinic acid of 1:1 and the reaction was continued at room temperature for 12 h. After the reaction was completed, the resulting polymer solution was introduced into a dialysis bag (8~14 kDa) and immersed in a glass container containing deionized water for dialyzing. Finally, the chitosan derivative was obtained by freeze drying.

[0027] Example 6 A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, modified zein prepared in Example 4, and chitosan derivative prepared in Example 5 were added to deionized water at 50°C to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:3 and then subjected to ultrasonic treatment for 35 minutes at a power of 150W and a temperature of 60°C. After drying, deer myocardial peptide microcapsules were obtained.

[0028] Example 7 A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, modified zein prepared in Example 4, and chitosan derivative prepared in Example 5 were added to deionized water at 50°C to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:5 and then subjected to ultrasonic treatment for 40 minutes at a power of 125W and a temperature of 60°C. After drying, deer myocardial peptide microcapsules were obtained.

[0029] Example 8 A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, modified zein prepared in Example 4, and chitosan derivative prepared in Example 5 were added to deionized water at 50°C to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:4 and then subjected to ultrasonic treatment for 30 minutes at a power of 175W and a temperature of 60°C. After drying, deer myocardial peptide microcapsules were obtained.

[0030] Example 9 A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, modified zein prepared in Example 4, and chitosan derivative prepared in Example 5 were added to deionized water at 50°C to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:4 and mixed. The mixture was then subjected to ultrasonic treatment for 35 minutes at a power of 125W and a temperature of 50°C. After drying, deer myocardial peptide microcapsules were obtained.

[0031] Example 10 A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, modified zein prepared in Example 4, and chitosan derivative prepared in Example 5 were added to deionized water at 50°C to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:4 and mixed. The mixture was then subjected to ultrasonic treatment for 60 minutes at a power of 150W and a temperature of 40°C. After drying, deer myocardial peptide microcapsules were obtained.

[0032] Comparative Example 1: A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, modified zein prepared in Example 4, and chitosan derivative prepared in Example 5 were added to deionized water at 50°C to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:4 and mixed. The mixture was then subjected to ultrasonic treatment for 20 minutes at a power of 125W and a temperature of 50°C. After drying, deer myocardial peptide microcapsules were obtained.

[0033] Comparative Example 2: A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, modified zein prepared in Example 4, and chitosan derivative prepared in Example 5 were added to deionized water at 50°C to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:4 and mixed. The mixture was then subjected to ultrasonic treatment for 35 minutes at a power of 125W and a temperature of 30°C. After drying, deer myocardial peptide microcapsules were obtained.

[0034] Comparative Example 3: A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, modified zein prepared in Example 4, and chitosan derivative prepared in Example 5 were added to deionized water at 50°C to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:4 and then subjected to ultrasonic treatment for 35 minutes at a power of 100W and a temperature of 50°C. After drying, deer myocardial peptide microcapsules were obtained.

[0035] Comparative Example 4: A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, zein prepared in Example 4, and chitosan derivative prepared in Example 5 were added to deionized water at 50°C in a mass ratio of 1:1:1 to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:4 and then subjected to ultrasonic treatment for 35 minutes at a power of 125W and a temperature of 50°C. After drying, deer myocardial peptide microcapsules were obtained.

[0036] Comparative Example 5: A method for preparing deer cardiac muscle peptide microcapsules, comprising the following steps: Resistant dextrin, modified zein prepared in Example 4, and chitosan were added to deionized water at 50°C at a mass ratio of 1:1:1 to prepare a wall material solution with a mass concentration of 10%. The solution was swollen in a water bath at 50°C for 30 minutes. 0.5g of deer myocardial peptide prepared in Example 1 was added to the wall material solution at a core-to-wall ratio of 1:4 and then subjected to ultrasonic treatment for 35 minutes at a power of 125W and a temperature of 50°C. After drying, deer myocardial peptide microcapsules were obtained.

[0037] Performance testing A. The deer heart muscle peptide prepared in Example 1 was used to prepare a deer heart enzymatic hydrolysate solution with a peptide concentration of 10 mg / mL. The effects of temperature, pH, food additives (sucrose, glucose and honey), preservatives (potassium sorbate, sodium benzoate), metal ions (KCl, CaCl2, ZnCl2, MgCl2, FeCl2), drying method (vacuum freeze drying, spray drying), in vitro gastrointestinal digestion, and storage conditions were determined. The hydroxyl radical scavenging rate was used as the indicator. All tests were repeated 3 times.

[0038] (1) Effect of different temperatures on deer myocardial peptides: After water bath heating treatment at 25℃, 40℃, 60℃, 80℃ and 100℃ for 1h, the hydroxyl radical scavenging rate was used as the indicator.

[0039] As can be seen from the table above, the deer cardiac muscle peptide prepared by this invention has good heat resistance.

[0040] (2) Effect of different pH values ​​on deer myocardial peptides: The pH was adjusted to different values ​​(2.0, 4.0, 6.0, 8.0, 10.0 and 12.0) by unidirectional adjustment with 1 mol / L HCl or 1 mol / L NaOH. After standing at room temperature (25℃) for 1 h, the pH was adjusted back to 7.0 and diluted to 1 mg / mL. The scavenging rate of hydroxyl radicals was used as the indicator.

[0041]

[0042] As shown in the table above, deer cardiac peptides have poor stability under excessively acidic and alkaline conditions.

[0043] (3) Effect of different concentrations of food additives on deer myocardial peptides: Different concentrations of sucrose, glucose and honey (2, 4, 6, 8, 10%) were added and mixed, and different concentrations of NaCl (0.5, 1.0, 1.5, 2.0, 2.5%) were added and mixed. After standing at room temperature (25℃) for 1 hour, the hydroxyl radical scavenging rate was used as the indicator.

[0044] ①The effect of different concentrations of NaCl added on deer cardiac muscle peptides:

[0045] ②The effects of different concentrations of sucrose, glucose, and honey added on deer cardiac muscle peptides:

[0046] As shown in the table above, excessive addition of NaCl, sucrose, glucose and honey to deer cardiac muscle peptides will affect their antioxidant properties to some extent.

[0047] (4) Effect of different concentrations of preservatives on deer myocardial peptides: Potassium sorbate (0.01, 0.02, 0.03, 0.04, 0.05%) and sodium benzoate (0.004, 0.008, 0.012, 0.016, 0.020%) were added and mixed, and then placed at room temperature (25℃) for 1 h. The scavenging rate of hydroxyl radicals was used as the indicator.

[0048] ①The effect of different concentrations of potassium sorbate added on deer cardiac muscle peptides:

[0049] ②The effect of different concentrations of sodium benzoate added on deer cardiac muscle peptides:

[0050] As shown in the table above, in practical applications, if preservatives need to be added, their negative impact on the stability of deer cardiac muscle peptides should be fully considered, and the amount used should be strictly controlled.

[0051] (5) Effect of different concentrations of metal ions on deer myocardial peptide: KCl, CaCl2, ZnCl2, MgCl2 and FeCl2 (0, 0.025, 0.05, 0.25, 0.5 and 1 mmol / L) were added and mixed, and then placed at room temperature (25℃) for 1 h. The scavenging rate of hydroxyl radicals was used as the indicator.

[0052]

[0053] As shown in the table above, when producing and storing deer cardiac muscle peptides, care should be taken to avoid contact with metal ions that affect stability, so as to prevent a decrease in antioxidant activity.

[0054] (6) Effect of different drying methods on deer heart muscle peptides: The deer heart enzymatic hydrolysate was dried by vacuum freeze drying and spray drying respectively, and the antioxidant activity of the product was measured, with the hydroxyl radical scavenging rate as the indicator.

[0055]

[0056] As shown in the table above, freeze drying is more effective in maintaining the antioxidant activity of deer cardiac muscle peptides because the low temperature during the freeze drying process reduces the thermal denaturation and oxidation of deer cardiac muscle peptides.

[0057] (7) Effect of in vitro gastrointestinal digestion on deer myocardial peptides: Deer myocardial peptides were dissolved in distilled water (3%, w / v), and the pH was adjusted to 2.0 with 1 mol / L HCl. 4% pepsin (w / w) was added, and the mixture was placed in a constant temperature shaker at 37℃ for 2 h to simulate gastric digestion. Then, the pH of the sample was adjusted to 5.3 with 0.9 mol / L NaHCO3, and then to 7.5 with 1 mol / L NaOH. 4% pancreatin (w / w) was added, and the mixture was placed in a constant temperature shaker at 37℃ for 2 h to simulate intestinal digestion. After digestion, the sample was placed in a boiling water bath for 10 min to inactivate the enzymes, and then cooled to room temperature. The sample after gastric digestion (GDs) and gastrointestinal digestion (GIDs) was centrifuged at 8000g and 4℃ for 10 min. The supernatant was collected, freeze-dried, and the sample was reconstituted to 1 mg / mL. Its activity was measured, with the hydroxyl radical scavenging rate as the indicator.

[0058]

[0059] As shown in the table above, the antioxidant activity of deer cardiac peptides gradually decreases during gastrointestinal digestion, but ultimately still retains a certain level of activity.

[0060] B. Protective effect of deer cardiac peptides against H2O2-induced cardiomyocyte damage: (1) Model preparation: H9C2 cardiomyocytes in the logarithmic growth phase were cultured with different concentrations of H2O2, and the cell viability was determined by MTT assay. A suitable H2O2 concentration was selected to prepare a cardiomyocyte oxidative damage model.

[0061] Please see Figure 1 The MTT assay results showed that different concentrations of H2O2 applied to cardiomyocytes had significant differences compared to the NC group (P<0.05), and significantly reduced cell survival. When the H2O2 concentration was 6 mmol / L, the cell survival rate was 52.80%, which was close to the half-inhibitory concentration. Therefore, this concentration was selected to create a cardiomyocyte oxidative stress model.

[0062] (2) Cell proliferation experiment: Cells were cultured with different concentrations of deer cardiac peptide, and the cell survival rate was determined by MTT assay after cell culture.

[0063] Please see Figure 2 Different concentrations of deer cardiac peptide were applied to cells, all of which promoted cell proliferation. Compared with the NC group, the concentrations of deer cardiac peptide at 0.3 mg / mL and 0.6 mg / mL significantly promoted cell proliferation (P<0.05). Therefore, three concentrations of 0.3 mg / mL, 0.6 mg / mL, and 0.9 mg / mL were selected for subsequent experiments.

[0064] (3) Pretreatment experiment: The effect of deer myocardial peptide pretreatment on H2O2-induced cardiomyocyte viability was determined.

[0065] Please see Figure 3 Compared with the NC group, the H2O2 model group significantly reduced cardiomyocyte survival rate (P<0.05), causing cardiomyocyte damage. However, the addition of deer cardiac peptide pretreatment significantly improved cardiomyocyte survival rate (P<0.05) in a concentration-dependent manner, indicating that deer cardiac peptide can significantly improve H2O2-induced cardiomyocyte damage.

[0066] (4) MDA / SOD content determination: The MDA / SOD content in cardiomyocytes was determined using a kit.

[0067] Please see Figure 4 Compared with the NC group, the H2O2 group showed a significant increase in MDA content (P<0.05) and a significant decrease in SOD content (P<0.05), indicating that H2O2 caused oxidative damage to cardiomyocytes. Compared with H2O2, the deer cardiac peptide pretreatment group significantly reduced MDA content (P<0.05) and enhanced SOD activity (P<0.05), indicating that deer cardiac peptide can reduce free radical attack on cardiomyocytes, reduce cardiomyocyte damage, enhance the cell's ability to scavenge free radicals, and thus enhance the cell's antioxidant capacity.

[0068] (5) Determination of AST, CK and LDH content: The content of the three myocardial enzymes, CK, AST and LDH, was detected according to the kit instructions.

[0069] Please see Figure 5 Compared with the NC group, the levels of CK, AST, and LDH in the cell supernatant of the H2O2 group were significantly increased (P<0.05), indicating that H2O2 caused cardiomyocyte damage. After pretreatment with deer cardiac peptide, the levels of myocardial enzymes in all groups significantly decreased (P<0.05). Therefore, deer cardiac peptide can reduce the levels of CK, AST, and LDH in H9C2 cardiomyocytes and has a protective effect against H2O2-induced H9C2 cell damage in a concentration-dependent manner.

[0070] (6) AO staining assay: apoptosis and necrosis of cells were determined by acridine orange (AO) staining. Apoptotic cells were observed to have dense and dark yellow-green staining under a fluorescence microscope.

[0071] Please see Figure 6 AO can cross the cell membrane and bind to nuclear DNA. Through repeated experiments (n≥3), it was found that cardiomyocytes shrank, became rounder and brighter after being treated with H2O2, indicating that H2O2 caused cardiomyocyte apoptosis. Pretreatment with deer cardiac peptide can reduce the degree of cell shrinkage and weaken the brightness of cardiomyocytes, indicating that deer cardiac peptide can improve the apoptosis of cardiomyocytes induced by H2O2.

[0072] (7) ROS content determination: The intracellular ROS content was determined using the DCFH-DA probe detection kit.

[0073] Please see Figure 7 The cells in the H2O2 group were the brightest, indicating a high intracellular ROS content and causing oxidative stress damage to the cells. The cells in the NC group and the deer cardiac peptide pretreatment group were significantly weaker than those in the H2O2-only treatment group. Therefore, pretreatment of cardiomyocytes with deer cardiac peptide can reduce the content of reactive oxygen species induced by H2O2 in cardiomyocytes and improve cardiomyocyte damage.

[0074] (8) The prepared deer cardiac muscle peptides were detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS), containing 2319 peptides. They were screened stepwise through an online database (biological activity, toxicity, physicochemical properties, antioxidant activity, hydrophobicity, ADMET). Molecular docking results revealed that four peptides (DWRPPRQ, WHEP, RHPYF, and NGPDHWHE) had outstanding binding ability. The amino acid sequence of DWRPPRQ is shown in SEQ ID NO 1, the amino acid sequence of WHEP is shown in SEQ ID NO 2, the amino acid sequence of RHPYF is shown in SEQ ID NO 3, and the amino acid sequence of NGPDHWHE is shown in SEQ ID NO 4. They mainly bind to free radicals (ABTS and DPPH), antioxidant enzymes (CAT and SOD), and antioxidant-related proteins (Keap-1) through hydrogen bonds, and have outstanding antioxidant capacity.

[0075] C. Immunomodulatory effects of deer cardiac peptide on RAW264.7 macrophages: (1) CCK8 toxicity test: The effect of different concentrations of deer myocardial peptide on cell survival rate was detected, and the appropriate concentration of deer myocardial peptide was screened for subsequent testing.

[0076] Please see Figure 8 The effect of different concentrations of deer cardiac peptide on the survival rate of RAW264.7 cells was investigated. Different letters indicate significant differences between them. Concentrations of 0.3, 0.5, 0.7, 0.9, 1.1, and 1.5 showed significant differences from the NC group (P<0.05), indicating that these concentrations of small molecule peptides can significantly promote cell proliferation. At the same time, there were inter-group differences among 0.1, 0.5, and 0.9 (P<0.05). Therefore, these three concentrations were selected as low, medium, and high concentrations for subsequent experiments.

[0077] (2) MDA content determination: The MDA content in RAW264.7 cells treated with different concentrations of deer myocardial peptide was detected using an MDA kit.

[0078] Please see Figure 9The body produces free radicals that react with lipids to generate MDA. MDA participates in the formation of lipid free radicals and oxygen uptake, and is a marker of endogenous lipid peroxides. Measuring the amount of MDA can indirectly reflect the degree of cell damage. Figure 9 The results showed that deer cardiac peptide had no significant effect on MDA content, indicating that deer cardiac peptide had no significant oxidative damage effect on RAW264.7 cells.

[0079] (3) NO / cytokine secretion detection: The immune activation effect was analyzed using an ELISA kit, and the secretion of NO, TNF-α, IL-1β and IL-6 in the culture supernatant of RAW264.7 cells after treatment with different concentrations of deer heart myocardial peptide was detected.

[0080] Please see Figure 10 NO (nitric oxide) is a key signaling molecule secreted by immune cells such as macrophages, and its biological activity is closely related to immune defense. NO exerts antibacterial, antiviral, and antitumor effects by damaging pathogen DNA and inhibiting enzyme activity, and is an important mediator of the innate immune response. Figure 10 It can be seen that the NO content in the blank group was much lower than that in the LPS group and the deer myocardial peptide group. After the treatment with deer myocardial peptide, the production of NO increased significantly and in a concentration-dependent manner. There were significant differences between the groups, which proves that deer myocardial peptide can simulate the immune activation effect of LPS in a dose-dependent manner.

[0081] Please see Figure 11 After treatment with different concentrations of deer heart myocardial peptide, the secretion of TNF-α, IL-1β, and IL-6 in the culture supernatant of RAW264.7 cells increased with increasing deer heart myocardial peptide concentration, and this increase was concentration-dependent. The secretion of cytokines TNF-α, IL-1β, and IL-6 in the cell culture supernatant was significantly increased compared with the blank control group (P<0.05). It can be concluded that small molecule peptides can induce the production of cytokines, indicating that deer heart myocardial peptide is a potential immunostimulant.

[0082] (4) Phagocytic function test: The phagocytic index of the treated cells was determined by the neutral red phagocytic test. Macrophages are used to take up foreign pathogens and are one of the most important non-specific immune responses of the body. The enhancement of phagocytic capacity is a commonly used assessment indicator of macrophage activation.

[0083] Please see Figure 12 Compared with the control group, the phagocytic index of both the LPS group and the deer cardiac peptide group was significantly increased (P<0.05), and this increase was concentration-dependent. The strength of macrophage immune function is evaluated by phagocytic activity; therefore, deer cardiac peptide can significantly enhance macrophage immune function.

[0084] (5) ROS level determination: The effect of deer heart myocardial peptide on ROS production in macrophages was detected by DCFH-DA fluorescent probe method. ROS level is an important mediator for macrophages to perform phagocytosis and bactericidal functions, and ROS detection can reflect the degree of immune activation of cells.

[0085] Please see Figure 13 The intracellular fluorescence intensity in the LPS group was significantly higher than that in the control group, indicating that LPS promotes the generation of ROS in RAW264.7 cells. At concentrations of 0.5 mg / ml and 0.9 mg / ml, the fluorescence intensity was also significantly enhanced compared to the control group, indicating that venom peptides can promote ROS production, enhance the phagocytic function of macrophages, and strengthen the immune response by activating macrophages.

[0086] (6) Adhesion ability determination: The state of cells after crystal violet staining was observed under an inverted microscope. The adhesion ability of macrophages is one of their important functions in the field of immunology.

[0087] Please see Figure 14 Compared with the control group, LPS enhanced cell adhesion. Furthermore, treatment with different concentrations of deer cardiac peptide enhanced the adhesion function of RAW264.7 cells. This indicates that deer cardiac peptide can promote adhesion and has an immune-activating effect.

[0088] (7) Cell cycle detection: Flow cytometry is used to analyze the distribution of cell proliferation stages. The proportion of cell cycle distribution is generally considered to be the main parameter of cell survival, growth and proliferation, including G0 / 1 phase, S phase and G2 / M phase.

[0089] The cell cycle distribution of RAW264.7 cells was analyzed using flow cytometry. Figure 15 The diagram shows the peak patterns of G0 / 1, S, and G2 / M. The meaning of G0 / 1 phase is: the period when the cell synthesizes a large number of substances, preparing for DNA synthesis; the meaning of S phase is: the period from the beginning to the completion of DNA synthesis and histone synthesis; the meaning of G2 / M phase is: the interval between the end of DNA replication and the beginning of mitosis.

[0090] like Figure 16 As shown, data analysis using Modfit 5.0 revealed that treatment with different concentrations of deer cardiac peptide reduced the proportion of macrophages in the G0 / 1 phase and correspondingly increased the proportion in the S phase. The higher the proportion of cells in the S phase, the stronger the cell proliferation activity, indicating that deer cardiac peptide may promote cell proliferation by increasing the proportion of macrophages in the S phase.

[0091] D. Protective effect of deer cardiac peptides on myocardial injury in mice (1) Measurement of myocardial infarction area in mice: The infarction area was calculated using the TTC method, and the degree of myocardial infarction was measured by the percentage of necrotic area to the total area.

[0092]

[0093] Note: Compared with the blank group, ***p<0.001; compared with the model group, ###p<0.001; NC: normal control group; ISO: isoproterenol model group; DS: compound Danshen tablets positive control group; DMP-L: low-dose deer myocardial peptide group; DMP-M: medium-dose deer myocardial peptide group; DMP-H: high-dose deer myocardial peptide group.

[0094] As shown in the table above, the myocardial infarction was most severe in the model group mice. Compared with the model group, the myocardial infarction area was reduced in the positive control group and each deer myocardial peptide dose group mice. The high dose of deer myocardial peptide had the best effect on reducing myocardial infarction in mice (P<0.001).

[0095] (2) CK / LDH / AST activity assay: Take blood from mouse eyeballs and perform the assay according to the kit instructions to measure CK, LDH and AST activities.

[0096]

[0097] Note: Compared with the blank group, ***p<0.001; compared with the model group, #p<0.05, ##p<0.01, ###p<0.001; NC: normal control group; ISO: isoproterenol model group; DS: compound Danshen tablets positive control group; DMP-L: low-dose deer myocardial peptide group; DMP-M: medium-dose deer myocardial peptide group; DMP-H: high-dose deer myocardial peptide group.

[0098] As shown in the table above, compared with the blank group, the serum levels of CK, AST, and LDH in the model group were significantly increased; compared with the model group, the serum levels of CK, AST, and LDH in each concentration group of deer cardiac peptide were significantly decreased. Therefore, deer cardiac peptide can reduce serum levels of CK, AST, and LDH, and has an ameliorative effect on myocardial injury in mice.

[0099] (3) Determination of SOD activity and MDA content: Determine SOD activity and MDA content according to the kit instructions.

[0100]

[0101] Note: Compared with the blank group, ***p<0.001; compared with the model group, #p<0.05, ##p<0.01, ###p<0.001; NC: normal control group; ISO: isoproterenol model group; DS: compound Danshen tablets positive control group; DMP-L: low-dose deer myocardial peptide group; DMP-M: medium-dose deer myocardial peptide group; DMP-H: high-dose deer myocardial peptide group.

[0102] As shown in the table above, compared with the control group, the model group showed a significant increase in MDA content and a significant decrease in SOD content. After treatment with different concentrations of deer cardiac peptide, the serum MDA content was significantly lower than that of the model group, while the SOD content was significantly higher. This indicates that deer cardiac peptide can reduce the attack of free radicals on cells, increase SOD activity, and thus improve the ability to scavenge free radicals. This suggests that deer cardiac peptide improves acute myocardial ischemia by exerting an antioxidant effect.

[0103] (4) Assay of TNF-α / IL-6 / IL-1β content: Take blood from mouse eyeballs and perform the assay according to the kit requirements to determine the activity of TNF-α, IL-6 and IL-1β.

[0104]

[0105] Note: Compared with the blank group, ***p<0.001; compared with the model group, #p<0.05, ##p<0.01, ###p<0.001; NC: normal control group; ISO: isoproterenol model group; DS: compound Danshen tablets positive control group; DMP-L: low-dose deer myocardial peptide group; DMP-M: medium-dose deer myocardial peptide group; DMP-H: high-dose deer myocardial peptide group.

[0106] As shown in the table above, compared with the blank group, the serum levels of TNF-α, IL-6, and IL-1β in the model group mice were significantly increased. Compared with the model group, the expression levels of TNF-α, IL-6, and IL-1β in the positive control group and each dose group of deer cardiac peptide were decreased, leading to a reduction in the expression of inflammatory factors and alleviating the symptoms of myocardial ischemia. This indicates that deer cardiac peptide can improve acute myocardial ischemia by exerting anti-inflammatory effects.

[0107] E. The encapsulation efficiency of the microcapsules prepared in Examples 6-10 and Comparative Examples 1-5 was determined: 0.1 g of microcapsule sample was weighed into a Buchner funnel and filtered with 10 mL of cold water. The content of unencapsulated peptides in the filtered solution was determined using a BCA kit. The dried microcapsule sample was weighed to obtain the total peptide content. The encapsulation efficiency was calculated as (total peptides - unencapsulated peptides) / total peptides × 100%. Staphylococcus aureus, Bacillus subtilis, and Escherichia coli were selected to evaluate the antibacterial performance of the microcapsules by the diameter of the inhibition zone. The data results are shown in Table 1.

[0108] Table 1 Results of Sample Encapsulation Efficiency and Antibacterial Properties

[0109] As shown in the table above, the encapsulation rate of the microcapsules prepared in Examples 6-10 of the present invention was 70.81-78.80%. Among them, the ultrasonic time, ultrasonic temperature and ultrasonic power were changed in Comparative Examples 1-3, respectively; no modification treatment was performed on zein in Comparative Example 4; and no rosmarinic acid grafting modification was performed on chitosan in Comparative Example 5. The encapsulation rate and antibacterial properties of Comparative Examples 1-5 were found to be lower than those of Examples 6-10.

[0110] E. The antioxidant activity of the microcapsules prepared in Examples 6-10 and Comparative Examples 1-5 was determined using the free radical scavenging rate as an indicator. The data results are shown in Table 2.

[0111] Table 2 Results of antioxidant activity assay of samples

[0112] As shown in the table above, the microcapsules prepared in Examples 6-10 of the present invention have a higher free radical scavenging rate than those in Comparative Examples 1-5, indicating that using resistant dextrin, modified zein, and chitosan derivatives as composite wall materials to encapsulate deer cardiac peptides is beneficial to synergistically improve antioxidant activity.

[0113] As can be seen from Tables 1 and 2 above, this application includes Comparative Examples 1-5. In Comparative Example 1, compared to Example 9, the ultrasonic time is shortened to 20 minutes; in Comparative Example 2, compared to Example 9, the ultrasonic temperature is reduced to 30°C; and in Comparative Example 3, compared to Example 9, the ultrasonic power is changed to 100W. The measured embedding rates in Comparative Examples 1-3 are significantly lower than those in Examples 6-10, thus affecting the antibacterial properties and antioxidant activity of the product. Therefore, claim 6 in the statement claims that "the ultrasonic treatment time is 30-60 minutes, and the power is 125-100W." "75W, temperature 40~60℃", under these conditions, the microcapsules prepared in Examples 6-10 had an encapsulation rate of 70.81~78.80%, indicating good encapsulation properties. In Comparative Example 4, zein was not modified, and in Comparative Example 5, chitosan was not grafted with rosmarinic acid. The encapsulation rate, antibacterial properties, and antioxidant activities of Comparative Examples 4-5 were found to be lower than those of Examples 6-10. This indicates that the modified zein and chitosan derivatives prepared in this application, as composite wall materials, have a synergistic effect in improving antibacterial properties and antioxidant activities when used to encapsulate deer cardiac peptides.

[0114] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0115] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for preparing deer cardiac muscle peptide, characterized in that, Includes the following steps: (1) After thawing frozen deer heart, remove the fascia, heart tube and fat, cut into pieces, wash and mince into meat paste; (2) The minced meat was enzymatically hydrolyzed, enzymes were inactivated, centrifuged, the supernatant was collected and dried to prepare deer myocardial peptide; The proteases used in the enzymatic hydrolysis are papain and flavor enzyme, with a material-to-liquid mass ratio of 1:3~3.5 and an enzymatic hydrolysis time of 4~5 hours; The ratio of papain to flavor enzyme is 4~4.5‰: 2~2.5‰.

2. The method for preparing deer cardiac muscle peptide according to claim 1, characterized in that, The protein content after enzymatic hydrolysis was measured to be 3.83-3.94%, the protein recovery rate was 90.23-93.36%, and the molecular weight distribution of 202-3000 Da was 90.6-93.8%.

3. The method for preparing deer cardiac muscle peptide according to claim 1, characterized in that, The drying method is one of spray drying, freeze drying, or low-temperature vacuum belt drying.

4. A deer cardiac muscle peptide, characterized in that, Prepared by the preparation method according to any one of claims 1 to 3, the peptide sequence of the deer cardiac muscle peptide includes DWRPPRQ, WHEP, RHPYF and NGPDHWHE, the amino acid sequence of DWRPPRQ is shown in SEQ ID NO 1, the amino acid sequence of WHEP is shown in SEQ ID NO 2, the amino acid sequence of RHPYF is shown in SEQ ID NO 3, and the amino acid sequence of NGPDHWHE is shown in SEQ ID NO 4.

5. An application of the deer cardiac muscle peptide according to claim 4, characterized in that, The deer myocardial peptide and the composite wall material are mixed and then subjected to ultrasonic treatment. The composite wall material is then coated on the surface of the deer myocardial peptide and dried to form deer myocardial peptide microcapsules. The ratio of the core material to the composite wall material of the deer myocardial peptide microcapsule is 1:3~5; the ultrasonic treatment time is 30~60min, the power is 125~175W, and the temperature is 40~60℃.

6. The application of the deer cardiac muscle peptide according to claim 5, characterized in that, The composite wall material is a resistant dextrin and chitosan derivative.

7. The application of the deer cardiac muscle peptide according to claim 5, characterized in that, The composite wall material is composed of resistant dextrin, modified zein, and chitosan derivatives.

8. The application of the deer cardiac muscle peptide according to claim 7, characterized in that, The preparation method of the modified zein includes the following steps: (1) Corn gluten powder was treated with α-amylase and acetone to remove starch and decolorize. It was then extracted with 70% ethanol solution and centrifuged at 3950~4100 r / min for 20~30 min. The supernatant was collected and then extracted with ice water. The precipitate was freeze-dried to prepare corn gliadin. (2) Dissolve zein in 70% ethanol solution and then slowly add it to 5% glucose solution and stir to react. Place it in a water bath at 55-65℃ and sonicate for 5-8 minutes to prepare modified zein. The mass ratio of glucose to zein is 1:8~12; the stirring reaction time is 20~40 min; and the ultrasonic power is 400~500W.

9. The application of deer cardiac muscle peptide according to claim 6 or 7, characterized in that, The preparation method of the chitosan derivative includes the following steps: dissolving chitosan in an aqueous acetic acid solution, stirring magnetically until a clear and transparent solution is obtained, then adding ascorbic acid and hydrogen peroxide, and stirring continuously for 25-40 minutes under nitrogen protection, followed by adding rosmarinic acid and continuing the reaction at room temperature for 10-12 hours. After the reaction is completed, the resulting polymer solution is introduced into a dialysis bag and immersed in a glass container containing deionized water for dialyzing. Finally, the chitosan derivative is obtained by freeze drying. The molar ratio of chitosan repeating units to rosmarinic acid is 1:1; the concentration of ascorbic acid is 0.2~0.4 mmol / L; and the concentration of hydrogen peroxide is 600~1000 mmol / L.

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