CN112522355A

CN112522355A - Method for optimizing deer blood enzymolysis peptide process by response surface method and application thereof

Info

Publication number: CN112522355A
Application number: CN202011403418.9A
Authority: CN
Inventors: 张辉; 李春楠; 吴楠; 杜延佳; 李晶峰; 李志成; 边学峰; 吕金朋; 尹馨雪; 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: 2021-03-19
Anticipated expiration: 2040-12-04
Also published as: CN112522355B

Abstract

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

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 using a response surface method and a research on a protective effect of the method on an LPS-induced H9c2 rat myocardial cell heart failure model.

Background

16-17% of organic matters in deer blood, wherein hemoglobin is mainly used, hemoglobin is composed of heme and globin, globin has large molecular weight, and is not easy to be digested and absorbed by human body when directly eaten (application of a deer blood bioactive peptide in preparation of blood pressure reducing medicines and health products [ A ]. China Association of animal husbandry, eighth (2017) Collection of Cervus chinensis's Production [ C ]. 2017: 3). In addition, the product prepared by fresh deer blood greatly influences the sensory state of people, and the basic constituent units of the polypeptide and the protein are amino acids, and the polypeptide and the protein are not different from each other in terms of amino acid nutrition. However, polypeptides have a much smaller relative molecular mass than proteins and have physiological regulatory functions not found in some proteins. The bioactive peptide can be absorbed by organism in an intact form, and can exert biological regulation effect under the condition of trace or low concentration. Therefore, the peptide in the protein is released by utilizing the degradation technology, the utilization rate of the protein can be improved, the human body can quickly supplement nutrition and energy, tens of millions of proteins in the human body can be quickly synthesized, and various physiological activity effects can be exerted. (Elstvler, Jens Adler-Nissen. enzymic Hydrolysis of Food proteins. applied Science Publishers [ J ], 1984)

Deer blood is bore blood or antler blood of sika deer (Cervus nippon Temminck) or red deer (Cervus elaphus Linnaeus), and is a traditional rare Chinese medicine. In recent years, research and development of deer blood products are widely concerned, the main forms of the deer blood products at present are deer blood wine, deer blood oral liquid, deer blood chewable tablets and deer blood nourishing capsules, and the deer blood products are special medicinal and edible food, and products such as deer blood egg custard, deer blood bean curd, deer blood porridge and the like also appear on the market. Modern clinical research shows that the deer blood peptide has obvious curative effects on oxidation resistance, immunity improvement, aging resistance, tumor resistance and the like.

The heart failure is a common clinical disease and a frequently-occurring disease and is the final outcome of various heart diseases, and according to epidemiological investigation of various parts of the world, 3-5 people have heart failure to some extent in the total population on average per 100 people. Therefore, the research on its pathogenesis and preventive treatment is also a hot spot of medical research. At present, the research on the protective effect of the deer blood enzymolysis peptide on an LPS-induced H9c2 rat myocardial cell heart failure model has not been carried out.

Disclosure of Invention

The invention aims to provide a method for optimizing a deer blood enzymolysis peptide process by using a response surface method and application thereof, and aims 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 is not developed at present.

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

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

(1) taking a certain amount of fresh deer blood filtered by gauze, adding a certain amount of distilled water according to 5% of substrate concentration, 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 constant at a required value (9-11), inactivating the enzyme in a boiling water bath for 30min after the hydrolysis reaction is finished (4-6 h), rapidly cooling, centrifuging hydrolysate for 30min at 3800r/min, collecting supernatant, and freeze-drying for later use.

(2) Selecting four factors of pH value, enzymolysis time, enzymolysis temperature and enzyme amount to carry out a single-factor experiment in the step (1), determining the optimal levels of the pH value, the enzymolysis time, the enzymolysis temperature and the enzyme amount, taking the optimal point of the single-factor experiment as the center, respectively taking 1 horizontal value from top to bottom around the optimal point as the level of a response surface, respectively coding the levels to be-1, 0 and 1, and carrying out the four-factor response surface experiment according to the Bx-Benhnken design principle.

TNF-alpha, IL-6, IL-1 beta are pro-inflammatory factors that are induced by Lipopolysaccharides (LPS) to cause apoptosis of myocardial cells. LPS is used for inducing H9c2 rat myocardial cells to establish a heart failure model, an ELISA kit is used for detecting the content levels of TNF-alpha, IL-6 and IL-1 beta in culture supernatant, and the protective effect of deer blood enzymolysis peptide on the LPS-induced H9c2 rat myocardial cell heart failure model is evaluated by taking the capability of deer blood enzymolysis oligopeptide (DBP-5) with different mass concentrations for inhibiting the release of TNF-alpha, IL-6 and IL-1 beta as a screening index.

Inducing H9c2 rat myocardial cells by using 1 mu g/ml LPS to establish a heart failure model, and determining the influence of deer blood enzymolysis peptide with different molecular weights on the proliferation inhibition activity of the H9c2 rat myocardial cells by using a thiazole blue (MTT) experiment; the ELISA kit is used for detecting the levels of TNF-alpha, IL-6 and IL-1 beta of culture supernatant, the capability of deer blood enzymolysis oligopeptide (DBP-5) with different mass concentrations for inhibiting the release of TNF-alpha, IL-6 and IL-1 beta is used as a screening index, and the protective effect of deer blood enzymolysis peptide on an LPS-induced H9c2 rat myocardial cell heart failure model is evaluated.

And analyzing the response surface test result by adopting Design Expert 10.0. The determination experiment of the cell inflammatory factors adopts SPSS 17.0 software to perform significance analysis of data, and the data is metered to

Shown, the comparisons between groups were performed using t-test and one-way analysis of variance.

The content of lysine in the deer blood enzymolysis peptide is the highest and is 17.81 percent by adopting an amino acid analyzer, and the content of human essential amino acid in the deer blood enzymolysis peptide is 52.78 percent.

The method for optimizing the 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 process conditions of the deer blood enzymolysis peptide are determined, the hydrolysis degree of the deer blood during enzymolysis can reach 28.98%, and a research basis is provided for further development and application of the deer blood.

In order to verify the protective effect of the deer blood enzymolysis peptide on the LPS-induced H9c2 rat myocardial cell heart failure model, the experiment researches the influence of the deer blood enzymolysis peptide with different molecular weights on the proliferation inhibition of H9c2 rat myocardial cells and the influence of deer blood oligopeptide (DBP-5) on the levels of TNF-alpha, IL-6 and IL-1 beta released by the H9c2 rat myocardial cells. The results show that the deer blood enzymolysis peptides with different molecular weights have inhibition effect on the proliferation of myocardial cells of H9c2 rats, and the deer blood enzymolysis oligopeptide (DBP-5) with the molecular weight less than 1 has the maximum inhibition rate on the proliferation. When the mass concentration of the deer blood enzymolysis oligopeptide is 200 mu g/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 H9c2 rat myocardial cells are reduced to different degrees, when the mass concentration reaches 200 mu g/mL, the release amount of the 3 cell inflammatory factors reaches the minimum, and the deer blood enzymolysis peptide is further verified to have a protective effect on an LPS-induced H9c2 rat myocardial cell heart failure model.

Drawings

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

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

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

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

FIG. 5a is a graph of the response of pH versus time.

Figure 5b is a contour plot of pH versus time interaction.

FIG. 6a is a graph of the response of pH versus temperature interaction.

Figure 6b is a contour plot of pH versus temperature interaction.

FIG. 7a is a graph of the response of pH interaction with enzyme amount.

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

FIG. 8a is a graph of the response of time versus temperature interaction.

Fig. 8b is a line contour plot of time versus temperature interaction.

FIG. 9a is a graph of the response of time interaction with enzyme amount.

FIG. 9b is a line contour plot of time versus enzyme amount interaction.

FIG. 10a is a graph of the response of temperature interaction with enzyme amount.

FIG. 10b is a line contour diagram showing the interaction of temperature and enzyme amount.

FIG. 11 shows the effect of BFP-5 on TNF- α secretion from rat cardiomyocytes of H9c 2.

FIG. 12 shows the effect of BFP-5 on IL-6 secretion from rat cardiomyocytes of H9c 2.

FIG. 13 shows the effect of BFP-5 on IL-1. beta. secretion from rat cardiomyocytes of H9c 2.

Detailed Description

In order to better understand the purpose, structure and function of the present invention, the following describes the method for optimizing deer blood enzymolysis peptide process and its application in response surface method in detail with reference to the attached drawings.

Example 1:

taking a certain amount of fresh deer blood filtered by gauze, adding a certain amount of distilled water, and preparing into a solution with a substrate concentration of 5%. Placing the prepared solution in a constant-temperature water and a pan with the temperature of 52 ℃, using 1mol/L NaOH or HCl to maintain the pH constant at 10.4, then adding 4400U/g of alkaline protease, using 1mol/L NaOH or HCl to maintain the pH value at 10.4 all the time in the reaction process, and carrying out enzymolysis for 5 h. After enzymolysis, adjusting pH of the enzymolysis solution to neutral, placing in boiling water bath for 30min, and inactivating enzyme. The obtained enzymolysis liquid is rapidly cooled, centrifuged for 30min at 3800r/min, the precipitate is discarded, and the supernatant is retained. Filtering the supernatant with filter membrane, and lyophilizing the filtrate to obtain sanguis Cervi enzymolysis peptide lyophilized powder.

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

(1) Measuring the degree of hydrolysis by a ninhydrin colorimetric method;

(2) experimental design and statistical analysis:

A. single factor test;

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

as can be seen from FIG. 1, the hydrolysis degree gradually increases with the gradually increasing pH value within the range of pH 9-10, and when the pH value reaches 10, the maximum hydrolysis degree of the deer blood enzymolysis peptide reaches 26.40%. Within the pH range of 10-11.0, the degree of hydrolysis is gradually reduced. This is because the enzyme, which is one of the proteins, has a close relationship between the enzymatic reaction rate and the pH, and when the pH is within a certain optimum range, the enzyme activity is highest and the enzymatic reaction rate is highest, and when the pH deviates from the optimum, the natural conformation of the enzyme and the dissociation state of the enzyme and the substrate are affected, which in turn affects the stability of the enzyme, inhibits the activity of the enzyme, and even inactivates, so that the optimum pH for enzymatic hydrolysis is determined to be 10.

As can be seen from figure 2, within the range of enzymolysis time of 4-5 h, the hydrolysis degree of the deer blood enzymolysis peptide is increased along with the time extension, and after 5h, the hydrolysis degree begins to decrease, because the concentration of the enzymolysis product is increased due to the continuous increase of the reaction time when the combination of the active site of the enzyme and the substrate reaches saturation and the reaction tends to be balanced along with the progress of the reaction, so that the competitive inhibition effect becomes stronger, and the side reaction is easy to occur on the newly generated products such as small molecular peptides, amino acids and the like, so the enzymatic reaction begins to decrease, and therefore, the optimal time is selected to be 5 h.

As can be seen from FIG. 3, the degree of hydrolysis gradually increased with the temperature gradually increased within the range of 40 to 55 ℃ and reached a maximum of 26.57% at 55 ℃. After the temperature is 55 ℃, the increase rate of the hydrolysis degree of the deer blood enzymolysis peptide is slowed down, because the activity of enzyme is enhanced along with the increase of the 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 the protein denaturation, the aggregation and precipitation of the protein are caused, the solubility of the protein is reduced, and the enzymolysis rate is reduced, so the enzymolysis temperature is preferably determined to be 55 ℃.

As can be seen from FIG. 4, the degree of hydrolysis of the peptides produced by enzymatic hydrolysis of deer blood gradually increased with the increase of the enzyme amount, and when the enzyme amount reached 4000U/g, the rate of increase of the degree of hydrolysis became smaller and even tended to decrease. This is because the substrate is saturated, the reaction rate is reduced due to the deterioration of the fluidity of the system, and the enzyme dosage is 4000U/g.

B. Response surface optimization test method

According to the single-factor test result, four factors of pH (A), time (B), temperature (C) and enzyme amount (D) are selected as optimization objects, and a four-factor three-level response surface test design is carried out. Fitting a quadratic polynomial model of nonlinear regression with experimental design software, the predicted model is 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

As can be seen from Table 3, the model is very significant (p <0.001), and the mismatching terms are not significant (p >0.05), indicating that the equation has no mismatching factors, and the regression model can be well fitted with the actual value. The influence sequence of 4 factors on the effect of the deer blood enzymolysis peptide obtained by the F test is as follows: pH (A) > enzyme amount (B) > temperature (C) > time (D). AC. The p-value of BC, CD is <0.05, which shows that the interaction between pH and temperature, enzyme amount and temperature, and enzyme amount and time has a significant effect on the degree of hydrolysis, and the p-value of BD, A2, B2, C2, D2 is <0.01, which is a very significant level. The decision coefficient R2 of the model is 0.9329, which shows that the regression equation obtained by response surface experimental design is better fitted 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

According to the regression equation analysis result, a response surface graph and a contour graph are made, the influence of the four factors of enzymolysis pH, time, temperature and enzyme amount on the enzymolysis effect of the deer blood peptidase is further visually confirmed, and the result is shown in figures 5 to 10.

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

Adopting Design Expert 10 software to analyze, and obtaining the optimal enzymolysis conditions as follows: pH is 10.43, enzymolysis time is 4.93h, enzymolysis temperature is 51.94 ℃, and enzyme amount is 4449.60U/g. The degree of hydrolysis predicted was 29.75%. Considering the possibility of practical operation, the experimental conditions were modified to: pH is 10.4, enzymolysis time is 5h, enzymolysis temperature is 52 ℃, and enzyme amount is 4400U/g. 3 replicates were run under this modified condition with an average value of 28.98%, close to the theoretical value, indicating that the mathematical model can be used to optimize the deer blood peptidase hydrolysis process.

Example 3: an H9c2 rat myocardial cell heart failure model is established by LPS induction, and the protective effect of deer blood enzymolysis peptide on the model is researched. 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 into a solution with a substrate concentration of 5%. Placing the prepared solution in a constant-temperature water bath kettle at the temperature of 52 ℃, using 1mol/L NaOH or HCl to maintain the pH constant at 10.4, then adding 4400U/g of alkaline protease, using 1mol/L NaOH or HCl to maintain the pH value at 10.4 all the time in the reaction process, and carrying out enzymolysis for 5 h. After enzymolysis, adjusting pH of the enzymolysis solution to neutral, placing in boiling water bath for 30min, and inactivating enzyme. The obtained enzymolysis liquid is rapidly cooled, centrifuged for 30min at 3800r/min, the precipitate is discarded, and the supernatant is retained.

(2) Microfiltering the supernatant obtained in the step (1) through a 0.45-micrometer filter membrane, ultrafiltering through a 1KDa, 3KDa and 10KDa ultrafiltration membrane, and obtaining a molecular total extract (DBP-1), a deer blood protein (DBP-2) with the mass of more than 10KDa, a deer blood polypeptide (DBP-3) with the molecular mass of 3KDa to 10KDa, a deer fetus polypeptide (DBP-4) with the molecular mass of 1KDa to 3KDa and a deer blood oligopeptide (DBP-5) with the molecular mass of less than 1KDa by adopting an ultrafiltration separation and purification method, and freeze-drying for later use.

(3) Cells in logarithmic growth phase were taken and plated. Discarding the culture solution, washing the cells for 2-3 times by using 3mL of PBS, discarding the PBS, adding a proper amount of cell digestion solution until the cells are completely digested, adjusting the density to 105/mL, inoculating 100 mu L of suspension cell solution per empty to a 96-hole culture plate, and adding 5% CO at 37 DEG C₂Culturing in an incubator for 24h, and performing grouping experiment when the growth reaches a monolayer fusion state. Adding 1 μ g/ml Lipopolysaccharide (LPS) to each group, and molding at 100 μ L except for blank control group, wherein the blank control group is added with 100 μ L DMEM (modified type) 10% fetal calf serum; after 24h incubation, 100. mu.L of the prepared sample solutions 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL were added to the sample group, and 100. mu.L of 10. mu.g/mL captopril was added to the positive control group, each of which had 5 duplicate wells. At 37 deg.C, 5% CO₂Culturing for 24h under the condition, discarding supernatant, adding 10 μ L of MTT (methanol to ethanol) 5mg/mL into each well, adding 100 μ L of DMSO after 4h, shaking for 5min, and measuring OD value at 490nm wavelength. And calculating the cell proliferation inhibition index. Cell proliferation inhibition ratio (%) [ (A blank control group-A administration group average)/A administration group average]X is 100%; table 4 shows the effect of deer blood zymolytic peptide on the inhibition of the proliferation of myocardial cells of H9c2 rats;

TABLE 4 measurement results of cell growth inhibition ratio in each group: (

n＝3)

Note: # indicates model vs blank, indicates dosing vs model group indicates P < 0.05; p < 0.01; p <0.001

Compared with a blank group, the cells of the LPS model group are remarkably proliferated (p is less than 0.001), and the success of the LPS model modeling is proved; the proliferation of cardiomyocytes in H9c2 rats was reduced to a different extent in each of the administered groups compared to the LPS model group, with the DBP-5 component being the most active. In the mass concentration range of 25-200 mug/mL, the cell proliferation inhibition index is increased along with the increase of the sample concentration and shows concentration dependence, when the concentration reaches 400 mug/mL, the cell proliferation inhibition index begins to be reduced, and each sample group has a significant difference (p <0.001) compared with the model group. The inhibition effect of the deer blood enzymolysis peptide is most obvious under the mass concentration of 200 mu g/mL. In conclusion, the results of the inhibitory activity of the deer blood zymolytic peptide on the proliferation of the H9c2 rat cardiac muscle cells are as follows: the component DBP-5 has the strongest activity under the mass concentration of 200 mug/mL.

(4) The cells in logarithmic growth phase were seeded in 96-well culture plates at a density of 105 cells/well in a volume of 200. mu.L/well at 37 ℃ with 5% CO₂Culturing for 24h in an incubator, after the cells adhere to the wall, discarding the supernatant, respectively adding 150 μ L of DBP-5 sample solution of 25, 50, 100, 200 and 400 μ g/mL, setting a blank group, a model group and a positive control group, and setting 5 multiple wells in each group. At 37 ℃ with 5% CO₂After 24h of culture under the condition, 150 mu L of supernatant is taken, and the secretion amounts of TNF-alpha, IL-6 and IL-1 beta are determined according to the kit instructions of TNF-alpha, IL-6 and IL-1 beta. FIGS. 11, 12 and 13 show the effect of deer blood zymolytic peptide on the release of TNF-alpha, IL-6 and IL-1 beta levels from rat cardiomyocytes;

from FIGS. 11-13, it is known that the mass concentration of deer blood enzymolysis oligopeptide is 200 μ g/mL, which has the strongest inhibitory effect on rat myocardial cells. After administration, the contents of TNF-alpha, IL-6 and IL-1 beta in the rat myocardial cell supernatant are reduced in different degrees, when the mass concentration reaches 200 mu g/mL, the release amount of the 3 cell inflammatory factors reaches the minimum, the protective effect of the deer blood enzymolysis peptide on a heart failure model is proved to be closely related to the release of the deer blood enzymolysis peptide for inhibiting the inflammatory factors, and the protection effect of the deer blood enzymolysis peptide on an LPS-induced H9c2 rat myocardial cell heart failure model is further verified.

Example 4: amino acid analysis of deer blood enzymolysis oligopeptide

Taking 10mg of lyophilized powder of deer blood oligopeptide (DBP-5) after enzymolysis, adding 10mL of 6mol/L hydrochloric acid to prepare a solution with the concentration of 1mg/mL, hydrolyzing at 110 ℃ for 24h, and determining the amino acid composition by an amino acid automatic detector, wherein the results are shown in Table 5.

TABLE 5 deer blood enzymolysis oligopeptide amino acid composition and content

As can be seen from Table 5, the deer blood enzymolysis peptide contains 17 amino acids, wherein the Lys content accounts for the highest percentage of the total content of the deer blood enzymolysis peptide amino acids, and accounts for 17.81%; the second is Phe and Ala, Gly, 14.97% and 10.32%, 8.68%, respectively. The content of essential amino acids (Lys, Phe, Val, Leu, Thr, Ile and Met) in human body is high and accounts for 52.78% of the total content of amino acids. Thr, Ile and Met are 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 indicates that deer blood has high nutritive value and health promotion function.

It is to be understood that the present invention has been described with reference to certain embodiments, and that various changes in the features and embodiments, or equivalent substitutions may be made therein 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. A method for optimizing a deer blood enzymolysis peptide process by a response surface method is characterized by comprising the following steps which are sequentially carried out:

step S1: taking a certain amount of fresh deer blood filtered by gauze, adding a certain amount of distilled water according to the concentration of 5% of a substrate, adding a certain amount of enzyme, hydrolyzing at a proper temperature, continuously adding 1mol/L NaOH or HCl in the reaction process to maintain the pH constant at a required value, inactivating the enzyme in a boiling water bath for 30min after the hydrolysis reaction is finished, rapidly cooling, centrifuging 3800r/min hydrolysate for 30min, collecting supernatant, and freeze-drying for later use;

step S2: selecting a plurality of factors to perform single-factor experiments on the step S1, determining each single factor corresponding to the optimal level, taking the optimal point of the single-factor experiment as the center, respectively taking 1 horizontal value around the optimal point as the level of the response surface, respectively coding-1, 0 and 1, and performing the factor response surface experiments according to a certain design principle.

2. The method for optimizing deer blood enzymolysis peptide process according to the response surface method of claim 1, further comprising the step of S3: a heart failure model is established by using LPS (lipopolysaccharide) with the concentration of 1 mu g/ml to induce H9c2 rat myocardial cells, and a thiazole blue experiment is used for determining the influence of the deer blood enzymolysis peptide with different molecular weights on the proliferation inhibition activity of the H9c2 rat myocardial cells.

3. The method for optimizing deer blood enzymolysis peptide process according to the response surface method of claim 2, further comprising the step of S4: analyzing the test result of the response surface by adopting Design Expert 10.0, analyzing the significance of data by adopting SPSS 17.0 software in the determination experiment of the cell inflammatory factors, and metering the data to

4. The method for optimizing deer blood enzymolysis peptide process according to the response surface method of claim 1, wherein the step S1: the use amount of the enzyme is 1000-5000U/g, the hydrolysis temperature is 40-60 ℃, the reaction is carried out for 4-6 h, and the pH is kept constant at 9-11.

5. The method for optimizing deer blood enzymolysis peptide process according to the response surface method of claim 1, wherein the step S2: selecting four factors of pH value, enzymolysis time, enzymolysis temperature and enzyme quantity to carry out single-factor experiment on the step S1, determining the optimal levels of the pH value, the enzymolysis time, the enzymolysis temperature and the enzyme quantity, taking the optimal point of the single-factor experiment as the center, respectively taking 1 horizontal value from top to bottom around the optimal point as the level of a response surface, respectively coding-1, 0 and 1, and carrying out the four-factor response surface experiment according to the Bx-Benhnken design principle.

6. The method for optimizing the deer blood enzymolysis peptide process according to the response surface method of claim 2, wherein the step S3 further comprises applying an ELISA kit to detect the levels of TNF- α, IL-6 and IL-1 β in the culture supernatant, and evaluating the protective effect of deer blood enzymolysis peptide on LPS-induced H9c2 rat myocardial cell heart failure model by using the ability of deer blood enzymolysis oligopeptide (DBP-5) with different mass concentrations to inhibit the release of TNF- α, IL-6 and IL-1 β as a screening index.

7. Use of the deer blood enzymolysis peptide prepared by the response surface method optimized deer blood enzymolysis peptide process according to any one of claims 1-6 in treating heart failure.