CN112522355B

CN112522355B - Method for optimizing deer blood enzymolysis peptide process by response surface method and application thereof

Info

Publication number: CN112522355B
Application number: CN202011403418.9A
Authority: CN
Inventors: 张辉; 尹馨雪; 李春楠; 吴楠; 杜延佳; 李晶峰; 李志成; 边学峰; 吕金朋; 兰梦; 高旭
Original assignee: Jilin Northeast Asia Biotechnology Co ltd
Current assignee: Jilin Northeast Asia Biotechnology Co ltd
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2023-06-02
Anticipated expiration: 2040-12-04
Also published as: CN112522355A

Abstract

The invention relates to a method for optimizing deer blood enzymolysis peptide technology by a response surface method and application thereof, belonging to the technical field of animal medicine extraction; according to the invention, through response surface optimization tests, the pH value, the extraction time, the extraction temperature and the extraction enzyme amount are respectively optimized, the optimal technological conditions of the deer blood enzymolysis peptide are determined, the hydrolysis degree of the deer blood enzymolysis peptide can reach 28.98%, a research foundation is provided for further development and application of the deer blood, the highest lysine content in the deer blood enzymolysis peptide is 17.81% and the necessary amino acid content of human body in the deer blood enzymolysis peptide is 52.78% by adopting an amino acid analyzer.

Description

Method for optimizing deer blood enzymolysis peptide process by response surface method and application thereof

Technical Field

The invention belongs to the technical field of animal medicine extraction, and particularly relates to a method for optimizing a deer blood enzymolysis peptide process by a response surface method and a research on protection effect of the deer blood enzymolysis peptide process on an LPS-induced H9c2 rat myocardial cell heart failure model.

Background

The organic matters in deer blood account for 16% -17%, wherein the organic matters are mainly hemoglobin, the hemoglobin consists of heme and globin, the molecular weight of the globin is large, and the globin is not easy to be digested and absorbed by human bodies when being directly eaten (Yan. The deer blood active peptide is applied to preparation of antihypertensive drugs and health care products [ A ]. Chinese animal husbandry society, eighth (2017) Chinese deer industry development society of Congress [ C ]. Chinese animal husbandry society: 2017:3). In addition, the product prepared by using fresh deer blood greatly influences the sensory state of people, and the basic constituent units of the polypeptide and the protein are amino acids, so that the product is analyzed from the aspect of amino acid nutrition, and the product and the protein are not different. However, the relative molecular mass of polypeptides is much smaller than that of proteins and has physiological regulatory functions that some proteins do not have. The bioactive peptide can be absorbed by organism in complete form, and can exert biological regulation effect under trace or low concentration condition. Therefore, the degradation technology is utilized to release the peptide in the protein, the protein utilization rate can be improved, the human body can rapidly supplement nutrition and energy, the human body tens of millions of proteins can be rapidly synthesized, and various physiological activities are exerted. (Elstvler, jens Adler-Nissen. Enzymatic Hydrolysis ofFood proteins, applied Science Publishers [ J ], 1984)

Deer blood is the bore blood or antler blood of sika deer (Cervus nipport Temminck) or red deer (Cervus elaphus Linnaeus), and is a traditional rare traditional Chinese medicine. In recent years, the development of deer blood products is widely focused, and the deer blood products are mainly in the forms of deer blood wine, deer blood oral liquid, deer blood chewing tablets, deer blood nourishing capsules, and are used as special medicine and food homologous foods, and products such as deer blood custard, deer blood bean curd, deer blood porridge and the like are also appeared on the market. Modern clinical researches show that deer blood peptide has remarkable curative effects in the aspects of antioxidation, immunity improvement, aging resistance, tumor resistance and the like.

Heart failure is a clinically common and frequently occurring disease, a final outcome of a variety of heart diseases, and epidemiological surveys throughout the world indicate that on average, every 100 people in the general population will have 3 to 5 people with some degree of heart failure. Thus, research on its pathogenesis and preventive treatment has also become a hotspot in medical research. At present, the protection effect of deer blood enzymolysis peptide on an LPS-induced H9c2 rat myocardial cell heart failure model has not been developed yet.

Disclosure of Invention

The invention aims to provide a method for optimizing a deer blood enzymolysis peptide process by a response surface method and application thereof, so as to solve the technical problems that the deer blood enzymolysis peptide process needs to be further optimized and the protection effect research of the deer blood enzymolysis peptide on an LPS-induced H9c2 rat myocardial cell heart failure model has not been developed yet.

In order to achieve the above purpose, the specific technical scheme of the method for optimizing deer blood enzymolysis peptide technology by using the response surface method and the application thereof is as follows:

the method for optimizing the deer blood enzymolysis peptide process by the response surface method comprises the following steps of:

(1) Taking a certain amount of fresh deer blood filtered by gauze, adding a certain amount of distilled water according to the substrate concentration of 5%, adding a certain amount of enzyme (1000-5000U/g), hydrolyzing at a proper temperature (40-60 ℃), continuously adding 1mol/L NaOH or HCl in the reaction process to maintain the pH value to be constant at a required value (9-11), inactivating enzyme in a boiling water bath for 30min after the hydrolysis (4-6 h) reaction is finished, rapidly cooling, centrifuging the hydrolysate 3800r/min for 30min, collecting supernatant and freeze-drying for later use.

(2) And (3) selecting four factors of pH value, enzymolysis time, enzymolysis temperature and enzyme amount to perform a single factor experiment on the step (1), determining the optimal level of the pH value, the enzymolysis time, the enzymolysis temperature and the enzyme amount, taking 1 level value around the optimal point of the single factor experiment as the level of a response surface respectively, encoding-1, 0 and 1 respectively, and performing a four factor response surface experiment according to a Bx-Benhnken design principle.

TNF- α, IL-6, IL-1β are pro-inflammatory factors that can be induced by Lipopolysaccharides (LPS) to cause myocardial apoptosis. The heart failure model is established by using LPS to induce H9c2 rat myocardial cells, ELISA kit is used for detecting the content level of TNF-alpha, IL-6 and IL-1 beta in culture supernatant, and the protection effect of deer blood enzymolysis oligopeptide (DBP-5) on the LPS-induced H9c2 rat myocardial cells heart failure model is evaluated by taking the capability of inhibiting TNF-alpha, IL-6 and IL-1 beta release of deer blood enzymolysis oligopeptide (DBP-5) with different mass concentrations as screening indexes.

Establishing a heart failure model by using 1 mug/ml LPS to induce H9c2 rat myocardial cells, and determining the influence of deer blood enzymolysis peptides with different molecular weights on the proliferation inhibition activity of the H9c2 rat myocardial cells by a thiazole blue (methyl thiazolyl tetrazolium, MTT) experiment; ELISA kit is used for detecting levels of culture supernatant TNF-alpha, IL-6 and IL-1 beta, and the protection effect of deer hematopoiesis oligopeptide (DBP-5) with different mass concentrations on the LPS-induced H9c2 rat myocardial cell heart failure model is evaluated by taking the ability of deer hematopoiesis oligopeptide (DBP-5) to inhibit release of TNF-alpha, IL-6 and IL-1 beta as screening indexes.

Response surface test results were analyzed using Design Expert 10.0. Cell inflammatory factor assay experiments data were analyzed for significance using SPSS 17.0 software, and data were measured to

The comparison between groups is shown using t-test and one-way analysis of variance.

The amino acid analyzer is adopted to obtain the deer blood enzymolysis peptide with the highest lysine content of 17.81 percent and the deer blood enzymolysis peptide with the higher human essential amino acid content of 52.78 percent.

The method for optimizing deer blood enzymolysis peptide process by using the response surface method and the application thereof have the following advantages: according to the invention, through a response surface optimization test, the extraction pH value, the extraction time, the extraction temperature and the extraction enzyme amount are respectively optimized, so that the optimal technological conditions of deer blood enzymolysis peptides are determined, the hydrolysis degree of deer blood can reach 28.98% during enzymolysis, and a research foundation is provided for further development and application of deer blood.

In order to verify the protection effect of deer blood enzymolysis peptide on LPS-induced H9c2 rat myocardial cell heart failure model, the experiment researches the influence of deer blood enzymolysis peptide with different molecular weights on proliferation inhibition of H9c2 rat myocardial cells and the influence of deer blood oligopeptide (DBP-5) on the level of releasing TNF-alpha, IL-6 and IL-1 beta from H9c2 rat myocardial cells. The results show that the deer blood enzymolysis peptides with different molecular weights have an inhibition effect on the proliferation of H9c2 rat myocardial cells, and the deer blood enzymolysis oligopeptide (DBP-5) with the molecular weight of less than 1 has the maximum proliferation inhibition rate. When the mass concentration of deer blood enzymolysis oligopeptide is 200 mug/mL, the inhibition effect on rat myocardial cells H9c2 is strongest. After administration, the contents of TNF-alpha, IL-6 and IL-1 beta in the supernatant of the myocardial cells of the H9c2 rat are reduced to different degrees, and when the mass concentration reaches 200 mug/mL, the release amount of the 3 cell inflammatory factors reaches the minimum, thereby further verifying that the deer hematolytic peptide has a protective effect on the LPS-induced heart failure model of the myocardial cells of the H9c2 rat.

Drawings

FIG. 1 shows the effect of different pH values on the degree of hydrolysis.

FIG. 2 shows the effect of different enzymolysis times on the degree of hydrolysis.

FIG. 3 shows the effect of different enzymatic hydrolysis temperatures on the degree of hydrolysis.

FIG. 4 shows the effect of different enzyme amounts on the degree of hydrolysis.

FIG. 5a is a response surface plot of pH versus time interactions.

FIG. 5b is a contour plot of pH versus time.

FIG. 6a is a response surface plot of pH versus temperature interactions.

FIG. 6b is a contour plot of pH versus temperature interactions.

FIG. 7a is a response surface plot of pH-enzyme interaction.

FIG. 7b is a contour plot of pH interaction with enzyme.

FIG. 8a is a response surface plot of time versus temperature interactions.

FIG. 8b is a contour plot of time versus temperature interactions.

FIG. 9a is a response surface plot of time-to-enzyme interactions.

FIG. 9b is a contour plot of time-enzyme interaction.

FIG. 10a is a response surface plot of temperature versus enzyme interaction.

FIG. 10b is a contour plot of temperature versus enzyme interaction.

FIG. 11 shows the effect of BFP-5 on TNF- α secretion from cardiomyocytes in H9c2 rats.

FIG. 12 is a graph showing the effect of BFP-5 on the IL-6 secretion of cardiomyocytes in H9c2 rats.

FIG. 13 is a graph showing the effect of BFP-5 on IL-1. Beta. Secretion from cardiomyocytes in H9c2 rats.

Detailed Description

In order to better understand the purpose, structure and function of the present invention, a method for optimizing deer blood enzymolysis peptide process by using the response surface method and application thereof are described in further detail below with reference to the accompanying drawings.

Example 1:

taking a certain amount of fresh deer blood filtered by gauze, adding a certain amount of distilled water, and preparing a solution with a substrate concentration of 5%. Placing the prepared solution into a constant temperature water bath kettle with the temperature of 52 ℃, maintaining the pH value to be constant at 10.4 by using 1mol/L NaOH or HCl, adding 4400U/g alkaline protease, and performing enzymolysis for 5h by using 1mol/L NaOH or HCl in the reaction process. After the enzymolysis is finished, the pH value of the enzymolysis liquid is adjusted to be neutral, and the enzymolysis liquid is placed in a boiling water bath for 30min to inactivate enzymes. The obtained enzymolysis liquid is rapidly cooled, centrifuged for 30min under 3800r/min, the precipitate is discarded, and the supernatant is reserved. Filtering the supernatant with a filter membrane, and lyophilizing the filtrate to obtain lyophilized powder of deer blood enzymolysis peptide.

Method for optimizing deer blood enzymolysis peptide process by response surface method

(1) Determining the degree of hydrolysis by using an ninhydrin colorimetric method;

(2) Experimental design and statistical analysis:

A. single factor test;

taking the degree of hydrolysis as an index, examining the influence of four factors of pH value 9-11, time 4-6 h, temperature 40-60 ℃ and enzyme quantity 1000U/g-5000U/g on the degree of hydrolysis of deer blood enzymolysis peptide, and the results are shown in figures 1-4:

as can be seen from FIG. 1, the hydrolysis degree gradually increases with increasing pH value in the range of 9-10, and the highest hydrolysis degree of deer blood enzymolysis peptide reaches 26.40% when the pH value reaches 10. In the pH range of 10 to 11.0, the degree of hydrolysis gradually decreases. The enzyme is used as one of proteins, the enzymatic reaction rate and the pH value are closely related, when the pH reaches a certain optimal range, the enzymatic activity reaches the highest, the enzymatic reaction rate is the highest, when the pH deviates from the optimal value, the natural conformation of the enzyme and the dissociation state of the enzyme and a substrate are influenced, so that the stability of the enzyme is influenced, the activity of the enzyme is inhibited and even inactivated, and the optimal pH for enzymolysis is determined to be 10.

As is clear from FIG. 2, in the range of 4 to 5 hours of the enzymolysis time, the hydrolysis degree of deer blood enzymolysis peptide increases with the increase of time, and after 5 hours, the hydrolysis degree starts to decrease, because as the reaction proceeds, when the active site of enzyme and the substrate are saturated, the reaction tends to be balanced, the concentration of the enzymolysis product increases due to the continuous increase of the reaction time, so that the competitive inhibition effect is enhanced, and the newly generated products such as small molecule peptide, amino acid and the like easily undergo side reaction, so that the enzymatic reaction starts to decrease, and therefore, the optimal time is selected to be 5 hours.

As can be seen from FIG. 3, the degree of hydrolysis gradually increases with increasing temperature in the range of 40 to 55℃and reaches a maximum of 26.57% at 55 ℃. The increase rate of the hydrolysis degree of deer blood enzymolysis peptide is reduced after 55 ℃, because the activity of enzyme is enhanced along with the increase of temperature in a certain temperature range, the enzymatic reaction is accelerated, when the enzymolysis temperature reaches a certain degree, the temperature is increased again to cause protein denaturation, aggregation and precipitation of the protein, and the solubility of the protein is reduced, so that the enzymolysis rate is reduced, and therefore, the enzymolysis temperature is determined to be preferably 55 ℃.

As is clear from FIG. 4, the degree of hydrolysis of deer blood enzymatic peptide gradually increases with increasing enzyme amount, and the rate of increase of the degree of hydrolysis becomes smaller and even decreases as the enzyme amount reaches 4000U/g. This is because the substrate is saturated, the fluidity of the system is deteriorated, the reaction rate is lowered, and 4000U/g is considered comprehensively.

B. Response surface optimization test method

According to the single-factor test result, four factors of pH (A), time (B), temperature (C) and enzyme quantity (D) are selected as optimization objects, and four-factor three-level response surface test design is carried out. Quadratic polynomial model fitting of nonlinear regression was performed using experimental design software, and the predicted model was as follows

Y＝31.50+1.28A+0.67B-1.86C+1.58D+0.56AB-2.32AC+0.24AD+2.34BC-2.76BD-2.31CD-5.80A2-5.26B2-4.29C2-4.01D2。

Specific test protocols are shown in table 1; the test results are shown in Table 2.

TABLE 1 factors and levels of response surface

TABLE 2 response surface optimization test design and results

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As can be seen from table 3, the model is extremely remarkable (p < 0.001), the mismatching term is not remarkable (p > 0.05), which indicates that the equation has no mismatching factor, and the regression model can be better fitted with the actual value. The F test can obtain the influence sequence of 4 factors on the deer blood enzymolysis peptide effect, which is as follows: pH (a) > enzyme amount (B) > temperature (C) > time (D). AC. The p-value of BC, CD <0.05, indicating that the interaction between pH and temperature, enzyme amount and time has a significant level of influence on the degree of hydrolysis, and the p-value of BD, A2, B2, C2, D2 <0.01, has an extremely significant level. The determination coefficient R2 = 0.9329 of the model shows that the regression equation obtained by the response surface test design fits well with the actual situation; the Coefficient of Variation (CV) was 0.7851%, indicating accurate and reliable experimental operation.

TABLE 3 analysis of variance and significance test of regression models

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And (3) making a response curve graph and a contour graph according to the analysis result of the regression equation, further intuitively confirming the influence of four factors of enzymolysis pH, time, temperature and enzyme quantity on the enzymolysis effect of deer blood peptide, and the results are shown in figures 5-10.

The contour lines are in the shape of ellipse, which indicates that the interaction of the factors is remarkable, while the circles indicate that the interaction is not remarkable, and as can be seen from fig. 5a and 5b, as the pH increases and the enzymolysis time increases, the hydrolysis degree increases and decreases, which indicates that there is a great point in the hydrolysis degree of deer blood enzymolysis peptide in this range, but no obvious interaction exists between factors AB. As can be seen from fig. 6a and 6b, the temperature is constant, and the degree of hydrolysis of the deer blood peptide gradually increases and then gradually decreases with increasing pH, which indicates that the maximum value of the degree of hydrolysis of the deer blood peptide exists in the range, and the contour lines are elliptical, which indicates that the interaction between the factors AC is remarkable; as can be seen from fig. 7a and 7b, the increase in pH and the increase in enzyme amount, the increase in hydrolysis degree and the decrease in hydrolysis degree, indicate that the maximum value exists in the hydrolysis degree of deer blood enzymolysis peptide in the range, and the interaction between factors AD is not obvious; as can be seen from fig. 8a and 8b, the temperature is constant, and the hydrolysis degree of deer blood enzymolysis peptide slowly increases and then starts to decrease with the extension of time, which indicates that the maximum value exists in the hydrolysis degree of deer blood enzymolysis peptide in the range, and the contour lines are elliptical, which indicates that the interaction between the factors BC is remarkable; as can be seen from fig. 9a and 9b, the time is constant, and the degree of hydrolysis of the deer blood peptide tends to gradually decrease after increasing with increasing enzyme amount, which indicates that there is an extreme value of the degree of hydrolysis of the deer blood peptide in the range, the contour line is elliptical, the curve trend is steepest, and the interaction between the factors BD is very remarkable; as can be seen from fig. 10a and 10b, the enzyme amount is constant, and the degree of hydrolysis of the deer blood enzymolysis peptide gradually increases and then decreases with increasing temperature, which indicates that the degree of hydrolysis of the deer blood enzymolysis peptide has a great point in the range; and the contour lines are oval, indicating significant interaction between factor CDs.

Analysis is carried out by adopting Design Expert 10 software, and the obtained optimal enzymolysis conditions are as follows: pH 10.43, enzymolysis time 4.93h, enzymolysis temperature 51.94 ℃ and enzyme amount 4449.60U/g. The predicted hydrolysis degree was 29.75%. The test conditions are modified to take into account the actual operational possibilities: pH 10.4, enzymolysis time 5h, enzymolysis temperature 52 ℃, enzyme amount 4400U/g. 3 parallel experiments are carried out under the correction condition, the average value is 28.98 percent, and the mathematical model is close to a theoretical value, so that the mathematical model can be used for optimizing the deer blood peptide enzymolysis process.

Example 3: the protection effect of deer hematopoiesis peptide on H9c2 rat myocardial cell heart failure model is researched by LPS induction. The method comprises the following steps:

(1) Taking a certain amount of fresh deer blood filtered by gauze, adding a certain amount of distilled water, and preparing a solution with a substrate concentration of 5%. Placing the prepared solution into a constant temperature water bath kettle with the temperature of 52 ℃, maintaining the pH value to be constant at 10.4 by using 1mol/L NaOH or HCl, adding 4400U/g alkaline protease, and performing enzymolysis for 5h by using 1mol/L NaOH or HCl in the reaction process. After the enzymolysis is finished, the pH value of the enzymolysis liquid is adjusted to be neutral, and the enzymolysis liquid is placed in a boiling water bath for 30min to inactivate enzymes. The obtained enzymolysis liquid is rapidly cooled, centrifuged for 30min under 3800r/min, the precipitate is discarded, and the supernatant is reserved.

(2) And (3) carrying out microfiltration on the supernatant obtained in the step (1) by using a 0.45 mu m filter membrane, carrying out ultrafiltration by using a 1kDa ultrafiltration membrane, a 3kDa ultrafiltration membrane and a 10kDa ultrafiltration membrane, obtaining a total molecular extract (DBP-1), deer blood protein (DBP-2) with the mass of more than 10kDa, deer blood polypeptide (DBP-3) with the mass of 3kDa to 10kDa, deer embryo polypeptide (DBP-4) with the mass of 1kDa to 3kDa and deer blood oligopeptide (DBP-5) with the mass of less than 1kDa by adopting an ultrafiltration separation and purification method, and freeze-drying for standby.

(3) Cells in logarithmic growth phase were taken and plated. Discarding culture solution, washing cells with 3mL PBS for 2-3 times, discarding PBS, adding appropriate amount of cell digestion solution, adjusting density to 105/mL, inoculating 100 μl of suspension cell solution per empty onto 96-well culture plate, and placing 5% CO at 37deg.C ₂ Culturing in an incubator for 24 hours, and performing a packet experiment when the strain grows to a single-layer fusion state. 100. Mu.L of Lipopolysaccharide (LPS) was added to each group for molding, except for the blank group, which was added with 100. Mu.L of DMEM (modified) 10% fetal bovine serum; after 24h incubation, 100. Mu.L of prepared 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL, 400. Mu.g/mL sample solution and cation were added to the sample setThe sex control group was 100. Mu.L of 10. Mu.g/mL captopril, 5 duplicate wells per group. At 37℃with 5% CO ₂ The culture was carried out for 24 hours, the supernatant was discarded, 5mg/mL MTT 10. Mu.L was added to each well, and after 4 hours, 100. Mu.L of DMSO was added thereto, and the mixture was shaken for 5 minutes to measure the OD at 490 nm. The cell proliferation inhibition index was calculated. Cell proliferation inhibition ratio (%) = [ (a blank group-a administration group average)/a administration group average]X 100%; table 4 shows the effect of deer blood enzymolysis peptides on inhibition of H9c2 rat myocardial cell proliferation;

TABLE 4 measurement results of cell proliferation inhibition ratios of the groups [ (]

n＝3)/>

Note that: # represents model vs. blank group, x represents dosing vs. model group, P <0.05; * Represents p < 0.01; * Represents p <0.001

Compared with a blank group, the LPS model group cells proliferate remarkably (p < 0.001), and the LPS model is proved to be successfully modeled; each of the dosing groups reduced proliferation of cardiomyocytes in H9c2 rats to a different extent than the LPS model group, with the DBP-5 component being the most active. In the mass concentration range of 25 to 200. Mu.g/mL, the cell proliferation inhibition index increased with increasing sample concentration, and showed concentration dependence, and when the concentration reached 400. Mu.g/mL, the cell proliferation inhibition index began to decrease, and the sample groups were significantly different from the model groups (p < 0.001). The inhibition effect of deer blood enzymolysis peptide is most remarkable at the mass concentration of 200 mug/mL. The inhibition activity of the deer blood enzymolysis peptide on the myocardial cell proliferation of the H9c2 rat is as follows: the component DBP-5 has the strongest activity at the mass concentration of 200 mug/mL.

(4) Inoculating logarithmic growth phase cells into 96-well culture plate with volume of 200 μl/well, and density of 200 μl/well105 cells/well at 37℃in 5% CO ₂ Culturing in incubator for 24 hr, removing supernatant after cell adhesion, adding 150 μl of DBP-5 sample solution of 25, 50, 100, 200, 400 μg/mL, and setting blank group, model group, positive control group, and 5 multiple wells. At 37℃with 5% CO ₂ After 24h of incubation, 150. Mu.L of supernatant was taken and the secretion amounts of TNF-. Alpha., IL-6, IL-1. Beta. Were measured according to the instructions of the TNF-. Alpha., IL-6, IL-1. Beta. Kit. FIGS. 11, 12, 13 are effects of deer hematopoietin on levels of TNF- α, IL-6, IL-1β released by rat cardiomyocytes;

from FIGS. 11 to 13, it was found that the inhibitory effect on rat myocardial cells was strongest at a mass concentration of 200. Mu.g/mL of deer blood enzymatic oligopeptide. After administration, the contents of TNF-alpha, IL-6 and IL-1 beta in rat myocardial cell supernatant are reduced to different degrees, and when the mass concentration reaches 200 mug/mL, the release amount of the 3 cell inflammatory factors reaches the minimum, which proves that the protection effect of deer blood enzymolysis peptide on heart failure models is closely related to the release of inflammatory factors inhibition by the deer blood enzymolysis peptide, and further verifies that the deer blood enzymolysis peptide has protection effect on LPS-induced H9c2 rat myocardial cell heart failure models.

Example 4: amino acid analysis of deer blood enzymatic oligopeptides

10mg of deer blood oligopeptide (DBP-5) freeze-dried powder after enzymolysis is taken, 10mL of 6mol/L hydrochloric acid is added to prepare a solution with the concentration of 1mg/mL, the solution is hydrolyzed for 24 hours at the temperature of 110 ℃, and the amino acid composition is measured by an amino acid automatic detector, and the result is shown in Table 5.

TABLE 5 deer blood enzymatic oligopeptide amino acid composition and content

As shown in Table 5, 17 amino acids were contained in the deer blood enzymolysis peptide, wherein the Lys content was the highest in the total amino acid content of the deer blood enzymolysis peptide, and was 17.81%; secondly, phe, ala and Gly respectively account for 14.97%, 10.32% and 8.68%. The content of the essential amino acid (Lys, phe, val, leu, thr, ile, met) of the human body is high, and the essential amino acid accounts for 52.78 percent of the total content of the amino acid. Thr, ile, met is lower than the essential amino acid pattern of the human body, and other essential amino acids are higher than or similar to the essential amino acid pattern of the human body. This shows that deer blood has high nutritive value and health care function.

It will be understood that the invention has been described in terms of several embodiments, and that various changes and equivalents may be made to these features and embodiments by those skilled in the art without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

1. The application of deer blood enzymolysis peptide in preparing medicaments for treating heart failure is characterized in that the preparation method of the deer blood enzymolysis peptide comprises the following steps:

taking a certain amount of fresh deer blood filtered by gauze, adding a certain amount of distilled water, and preparing a solution with a substrate concentration of 5%; placing the prepared solution in a constant temperature water bath kettle with the temperature of 52 ℃, maintaining the pH value to be 10.4 by using 1mol/L NaOH or HCl, adding 4400U/g alkaline protease, and performing enzymolysis for 5h by using 1mol/L NaOH or HCl in the reaction process; after the enzymolysis is finished, regulating the pH value of the enzymolysis liquid to be neutral, placing the enzymolysis liquid in a boiling water bath for 30min, and inactivating enzymes; the obtained enzymolysis liquid is rapidly cooled, centrifuged for 30min under 3800r/min, the sediment is discarded, and the supernatant is reserved; filtering the supernatant with a filter membrane, and lyophilizing the filtrate to obtain lyophilized powder of deer blood enzymolysis peptide.