Bifunctional peptide with ACE inhibitory activity and antioxidant activity, preparation method and application

Abstract

The present invention discloses a bifunctional peptide with ACE inhibitory and antioxidant activity, whose amino acid sequence is IHWPTGF, AAPF, or IGF. The present invention also discloses a method for preparing a donkey meat polypeptide containing the bifunctional peptide with ACE inhibitory and antioxidant activity, comprising: 1) heating raw donkey meat at a temperature of 45-95°C for 30-90 minutes, then stopping the thermal reaction; 2) subjecting the heated donkey meat to in vitro simulated gastric biomimetic enzymatic hydrolysis and in vitro simulated intestinal biomimetic enzymatic hydrolysis to obtain a donkey meat polypeptide solution. The present invention also discloses a method for preparing a donkey meat polypeptide health product, comprising: filtering the donkey meat polypeptide solution to obtain a filtrate, mixing the filtrate with a deodorizing agent to remove the odor, and then sterilizing and drying to obtain a donkey meat polypeptide powder. The present invention optimizes the optimal heat treatment process for donkey meat, clarifies the structure of bioactive peptides in donkey meat, and analyzes their active sites, obtaining novel peptides with stable functional activity. This results in the development of a solid polypeptide health product with ACE inhibitory and antioxidant activity, providing a scientific basis for the high-value utilization of donkey meat and the development of functional foods.

Description

Technical Field

[0001] The present invention belongs to the field of food technology, and specifically relates to a bifunctional peptide segment with ACE inhibitory activity and antioxidant activity, a preparation method and an application thereof. Background Art

[0002] With the rapid development of my country's economy, people's demand for meat has shifted from simply having meat to simply eating good meat, with a greater emphasis on improving the quality and structure of meat. This has led to a growing demand for high-quality donkey meat, and donkey meat products continue to outstrip supply. Donkey meat is rich in protein, amino acids, and unsaturated fatty acids, and is low in fat, cholesterol, and calories, making it suitable for a variety of populations, particularly those with hypertension, obesity, and cardiovascular and cerebrovascular diseases. Current research on donkey meat focuses primarily on quality identification, preservation, and traditional processing, with limited research on its advanced processing. Functional peptides derived from animal proteins have numerous physiological functions, including antihypertensive, antioxidant, hypoglycemic, and immune-enhancing properties, and have therefore become a research hotspot both domestically and internationally. However, reports on the preparation of functional peptides from donkey meat are limited, and the composition, structure, and active sites of these peptides remain largely unknown. There are also no donkey meat peptide-based products on the market. As a high-quality meat resource, donkey meat's peptide components may possess specific nutritional and health benefits, and exploring their functional activities is of great value to the food industry and public health. Summary of the Invention

[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0004] Another object of the present invention is to provide a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity.

[0005] Another object of the present invention is to provide a method for preparing a donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity.

[0006] Another object of the present invention is to provide a method for preparing a donkey meat polypeptide health product containing a bifunctional peptide segment.

[0007] To this end, the technical solution provided by the present invention is:

[0008] A bifunctional peptide segment with ACE inhibitory activity and antioxidant activity, wherein the amino acid sequence of the bifunctional peptide segment is IHWPTGF, AAPF or IGF.

[0009] A method for preparing a donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity comprises the following steps:

[0010] 1) heating the donkey meat raw material at a temperature of 45-95° C. for 30-90 minutes, and then stopping the thermal reaction;

[0011] 2) The heated donkey meat is subjected to in vitro simulated gastric biomimetic enzymolysis and in vitro simulated intestinal biomimetic enzymolysis in sequence to obtain a donkey meat polypeptide solution containing a bifunctional peptide segment with ACE inhibitory activity and antioxidant activity, wherein the amino acid sequence of the bifunctional peptide segment with ACE inhibitory activity and antioxidant activity is IHWPTGF, AAPF or IGF.

[0012] Preferably, in the method for preparing the donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity, in step 1), the donkey meat raw material is cut into blocks of 3×3×2 cm.

[0013] Preferably, in the method for preparing the donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity, in step 1), the donkey meat raw material is heated at 95° C. for 75 minutes.

[0014] Preferably, in the preparation method of the donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity, in step 2), the method of simulating in vitro gastric biomimetic enzymatic hydrolysis comprises: taking the heated donkey meat, mincing it and adding it to water, the mass volume ratio of donkey meat to water is 1:4, after homogenization, adjusting the pH value to 2.0±0.1, adding 40,000 U / g of pepsin accounting for 1% of the volume of the reaction system, and simulating in vitro gastric enzymatic hydrolysis for 2 hours at 37°C and a constant temperature vibration environment.

[0015] Preferably, in the preparation method of the donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity, in step 2), the method of simulating in vitro intestinal biomimetic enzymatic hydrolysis comprises: after performing the in vitro simulated gastric biomimetic enzymatic hydrolysis, adjusting the pH value of the enzymatic hydrolysis solution to 7.5±0.1, adding 40,000 U / g trypsin solution accounting for 1% of the volume of the reaction system, and simulating in vitro intestinal fluid enzymatic hydrolysis for 2 hours at 37°C and a constant temperature vibration environment.

[0016] A method for preparing a donkey meat polypeptide health product containing a bifunctional peptide segment comprises the following steps:

[0017] 1) heating the donkey meat raw material at a temperature of 45-95° C. for 30-90 minutes, and then stopping the thermal reaction;

[0018] 2) subjecting the heated donkey meat to in vitro simulated gastric biomimetic enzymolysis and in vitro simulated intestinal biomimetic enzymolysis in sequence to obtain a donkey meat polypeptide solution containing bifunctional peptides having ACE inhibitory activity and antioxidant activity;

[0019] 3) After the enzymatic hydrolysis is completed, the donkey meat polypeptide solution is filtered to obtain a filtrate, the filtrate is mixed with a deodorizing agent to perform a deodorizing treatment, and then sterilized and dried to obtain a donkey meat polypeptide powder, wherein the deodorizing treatment is performed by any one of the following methods:

[0020] Use 0.2% active dry yeast at a temperature of 35°C to remove the fishy smell for 60 minutes;

[0021] Use 2.0% β-cyclodextrin to remove the fishy smell at 60°C for 40 minutes; or

[0022] First, active dry yeast with a mass concentration of 0.2% was used to remove the fishy smell at a temperature of 35°C for 60 minutes, and then β-cyclodextrin with a mass concentration of 2.0% was used to remove the fishy smell at a temperature of 60°C for 40 minutes.

[0023] 4) The donkey meat polypeptide powder is formulated to obtain a donkey meat polypeptide health product.

[0024] Preferably, in the preparation method of the donkey meat polypeptide health product containing a bifunctional peptide segment, in step 4), citric acid, xylitol and lemon essence are used for preparation, and the mass ratio of the donkey meat polypeptide powder to citric acid, xylitol and lemon essence is: 20-30:1.5-2.5:60-100:1.5-2.5.

[0025] Preferably, in the method for preparing the donkey meat polypeptide health product containing the bifunctional peptide segment, the mass ratio of the donkey meat polypeptide powder to citric acid, xylitol and lemon essence is 25:2.5:60:2.

[0026] Preferably, in the method for preparing the donkey meat polypeptide health product comprising a bifunctional peptide segment, the donkey meat polypeptide is prepared by any of the methods described above, and the donkey meat polypeptide health product is prepared by any of the methods described above.

[0027] The present invention has at least the following beneficial effects:

[0028] The present invention optimizes the optimal heat treatment process for donkey meat, clarifies the structure of bioactive peptides in donkey meat and analyzes their active sites, obtains new peptide segments with stable functional activity, and develops a polypeptide solid health product with ACE inhibitory activity and antioxidant activity, providing a scientific basis for the high-value utilization of donkey meat and the development of functional foods.

[0029] Other advantages, objectives and features of the present invention will be reflected in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1This figure shows the ACE inhibitory activity of donkey meat peptides prepared under different cooking conditions according to the present invention. Note: Different capital letters in the figure indicate that the difference in ACE inhibitory activity between different heating temperatures at the same heating time reaches a significant level (P < 0.05). Different lowercase letters indicate the difference in ACE inhibitory activity between different heating times at the same heating temperature (P < 0.05).

[0031] Figure 2 The ABTS free radical scavenging rates of donkey meat peptides prepared under different cooking conditions according to the present invention are shown. Note: Different capital letters in the figure indicate that the difference in ABTS free radical scavenging rates between different heating temperatures at the same heating time is significant (P < 0.05). Different lowercase letters indicate that the difference in ABTS free radical scavenging rates between the same heating temperature at different heating times is significant (P < 0.05).

[0032] Figure 3 The DPPH radical scavenging rates of donkey meat peptides prepared under different cooking conditions according to the present invention are shown. Note: Different capital letters in the figure indicate that the difference in DPPH radical scavenging rates between different heating temperatures at the same heating time is significant (P < 0.05). Different lowercase letters indicate that the difference in DPPH radical scavenging rates between the same heating temperature at different heating times is significant (P < 0.05).

[0033] Figure 4 The hydroxyl radical scavenging rates of donkey meat peptides prepared under different cooking conditions according to the present invention are shown. Note: Different capital letters in the figure indicate that the difference in hydroxyl radical scavenging rates between different heating temperatures at the same heating time is significant (P < 0.05). Different lowercase letters indicate that the difference in hydroxyl radical scavenging rates between the same heating temperature at different heating times is significant (P < 0.05).

[0034] Figure 5 This figure shows the reducing ability of donkey meat peptides prepared under different cooking conditions according to the present invention. Note: Different capital letters in the figure indicate that the difference in reducing ability between different heating temperatures at the same heating time is significant (P < 0.05). Different lowercase letters indicate that the difference in reducing ability between the same heating temperature at different heating times is significant (P < 0.05).

[0035] Figure 6 The antioxidant activity (Y value) of donkey meat under different heating conditions in the examples of the present invention is shown.

[0036] Figure 7 Molecular docking of ACE inhibitory peptides in the examples of the present invention is shown: (A) IHWPTGF; (B) IGF; (C) AAPF.

[0037] Figure 8Molecular docking of the MPO inhibitory peptides in the examples of the present invention is shown: (A) IHWPTGF; (B) IGF; (C) AAPF.

[0038] Figure 9 This is a graph showing the effect of the amount of donkey meat polypeptide added on the sensory quality of solid health products in an embodiment of the present invention.

[0039] Figure 10 This is a graph showing the effect of citric acid addition on the sensory quality of solid health products in an embodiment of the present invention.

[0040] Figure 11 This is a graph showing the effect of xylitol addition on the sensory quality of solid health products in an embodiment of the present invention.

[0041] Figure 12 This is a graph showing the effect of the amount of lemon essence added on the sensory quality of solid health products in an embodiment of the present invention.

[0042] Figure 13 This is a flow chart of the preparation process of polypeptide health products in an embodiment of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0044] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0045] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0046] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following examples are provided for further explanation:

[0047] Example 1

[0048] 1.1 Cooking conditions

[0049] Thaw the donkey meat at 4°C and rinse with running water to remove any blood. Remove the surface fascia and fat, then cut into 3 × 3 × 2 cm blocks along the grain. Place the meat sample in a retort bag.

[0050] Place the cooking bag containing the sample into a water bath, and start timing when the center temperature of the meat reaches the set temperature. Heat at 45℃, 55℃, 65℃, 75℃, 85℃, and 95℃ for 30min, 45min, 60min, 75min, and 90min respectively. After heating, soak the cooking bag in ice water for 10min to stop the thermal reaction.

[0051] 1.2 In vitro simulated enzymatic hydrolysis

[0052] Simulated gastric hydrolysis experiment: Add 40 mL of distilled water to 10.00 g of minced donkey meat sample and homogenize five times at 8,000 rpm, each homogenization lasting 30 seconds. Adjust the pH to 2.0 ± 0.1. Add 20 mL of pepsin solution (40,000 U / g, 1%) to each sample, mix thoroughly, and simulate in vitro gastric hydrolysis at 37°C for 2 hours on a constant temperature shaker at 160 rpm.

[0053] Simulated Intestinal Hydrolysis Experiment: After simulated gastric hydrolysis, adjust the pH of the hydrolyzate to 7.5 ± 0.1 and add 20.0 mL of trypsin solution (40,000 U / g, 1%). Incubate the hydrolyzate at 37°C for 2 h in simulated intestinal fluid, with a constant temperature oscillator at 160 rpm. After the simulated intestinal hydrolysis, heat in a boiling water bath for 5 min to terminate the hydrolysis reaction.

[0054] 1.3 Preparation of donkey meat peptides

[0055] After the enzymatic hydrolysis, the mixed solution was centrifuged (4°C, 20 min), the supernatant was filtered with filter paper, the filtrate was passed through a 0.22 μm membrane, and freeze-dried for 48 h. The solid powder was collected and stored in the dark.

[0056] 1.4 ACE inhibitory activity assay

[0057] ACE inhibitory activity of donkey meat peptides prepared under different cooking conditions Figure 1 As shown in the figure, at the same temperature, the ACE inhibition rate showed an increase-decrease trend with prolonged heating time, but when heated at 65°C, it showed a trend of first increasing and then decreasing with prolonged heating time, and when heated at 75°C, the ACE inhibition activity first decreased and then increased.

[0058] Table 1 Analysis of the significance of ACE inhibitory activity of donkey meat peptides prepared under different cooking conditions

[0059]

[0060] At the same time, as the heating temperature increased, the ACE inhibition rate showed an increasing-decreasing trend, indicating that higher temperatures do not necessarily lead to better ACE inhibitory activity, and extreme heat treatment may reduce ACE inhibitory activity. When the heating time was 60 minutes, the ACE inhibition rate did not change significantly before 65°C. After 75°C, the inhibition rate decreased as the heating temperature increased. This may be due to the dissociation of actomyosin at 70°C, resulting in a decrease in the production of ACE inhibitory peptide fragments.

[0061] The 30 groups of samples were subjected to significant difference analysis (Table 1). The results showed that samples 13, 16, 29, 17, and 20 (65°C / 60 min, 75°C / 30 min, 95°C / 75 min, 75°C / 45 min, and 75°C / 90 min) had high ACE inhibitory activity and no significant differences were found.

[0062] 1.5 ABTS free radical scavenging rate determination

[0063] ABTS free radical scavenging rate of donkey meat cooked under different conditions Figure 2 As shown, the ABTS free radical scavenging rate showed a small change trend, generally decreasing first and then increasing. At the same heating time, the ABTS free radical scavenging rate first decreased and then increased with increasing heating temperature. At the same heating temperature, the relationship between the ABTS free radical scavenging rate and heating time was not obvious. This may be because the simulated enzymatic hydrolysis of donkey meat at different heating times in vitro releases peptides with similar ABTS free radical scavenging abilities.

[0064] 1.6 DPPH free radical scavenging rate determination

[0065] Figure 3 The DPPH radical scavenging rate of donkey meat peptides under different cooking conditions was analyzed. At the same temperature, the DPPH radical scavenging rate gradually increased with increasing heating time. When heated at 45°C, the scavenging rate decreased with increasing heating time. When heated at 65°C, the scavenging rate initially increased and then decreased, likely due to muscle protein denaturation. After 30 minutes of heating, the DPPH radical scavenging rate decreased with increasing heating temperature. After 90 minutes of heating, the scavenging rate showed an increasing-decreasing trend with increasing temperature.

[0066] 1.7 Hydroxyl radical scavenging rate determination

[0067] Hydroxyl radical scavenging rate ( Figure 4) showed an overall trend of first increasing and then decreasing with increasing heating time and temperature. When heated at 45°C and 55°C, the hydroxyl radical scavenging rate increased with time. The hydroxyl radical scavenging rate of samples cooked at 55°C was higher than that of samples cooked at 45°C. The scavenging rate in the 55°C / 90 min treatment group was the highest, reaching 72.71±1.80%. This may be because 55°C is the denaturation temperature of myosin, which produces more antioxidant peptide fragments.

[0068] 1.8 Reduction ability test

[0069] Reduction ability ( Figure 5 ) showed an overall trend of first decreasing and then increasing, which is consistent with the trend of changes in ABTS free radical scavenging rate. At the same heating time, the reducing capacity first decreased and then increased with increasing temperature. However, at a heating time of 90 minutes, the reducing capacity first increased and then decreased with increasing temperature. At 85°C, the reducing capacity increased, which may be related to the dissolution and gelation of collagen at high temperature.

[0070] 1.9 Determination of optimal cooking conditions

[0071] As shown in Table 2, a larger correlation coefficient among the four indicators indicates a higher correlation, and a smaller correlation coefficient indicates a lower correlation. The four indicators of ABTS free radical scavenging rate (X1), DPPH free radical scavenging rate (X2), hydroxyl free radical scavenging rate (X2), and reducing ability (X4) were selected for principal component analysis. Fewer indicators were used to replace more indicators while retaining the main information of the original indicators to select the optimal heating conditions for the antioxidant activity of donkey meat polypeptides.

[0072] Table 2 Correlation matrix between indicators

[0073]

[0074] Table 3 Correlation matrix eigenvalues

[0075]

[0076] Note: Extraction method: principal component analysis

[0077] Table 4 Eigenvectors of each principal component

[0078]

[0079] As shown in Table 3, the cumulative variance contribution rate of the first two principal components reached 69.33%, so the two principal components were selected to comprehensively analyze the heating conditions. According to the principal component eigenvectors in Table 4, the linear relationship between the principal components and the four antioxidant indicators of donkey meat peptides was constructed as follows:

[0080] Y=0.4109Y1+0.2825Y2

[0081] Y1=0.58168*X1+0.52138*X2-0.14926*X3+0.60624*X4

[0082] Y2=-0.17135*X1+0.49626*X2+0.84943*X3-0.05325*X4

[0083] After standardizing the four indicators, the comprehensive evaluation model Y is constructed with the ratio of the eigenvalues ​​of the two principal components Y1 and Y2 to the total eigenvalues ​​as the weight. Y is the linear combination of Y1 and Y2, that is: Y = 0.4109Y1 + 0.2825Y2. The comprehensive analysis results are as follows: Figure 6 shown.

[0084] Depend on Figure 6 It can be seen that the Y values ​​of the 55℃ / 90min, 85℃ / 75min, 95℃ / 45min, 95℃ / 90min, 55℃ / 45min, 95℃ / 75min, 65℃ / 45min, 75℃ / 90min, 85℃ / 90min, 45℃ / 30min, 45℃ / 75min, 65℃ / 90min, and 55℃ / 75min treatment groups are higher and greater than 30, indicating higher antioxidant activity.

[0085] Both the 95°C / 75 min and 75°C / 90 min treatment groups exhibited high antioxidant and ACE inhibitory activities, but the 95°C / 75 min treatment group exhibited superior antioxidant activity compared to the 75°C / 90 min treatment group. In actual processing applications, 95°C is within the boiling temperature range of water, making temperature control easier. Furthermore, the 95°C / 75 min treatment was more time-efficient. Therefore, overall, the 95°C / 75 min treatment group exhibited the highest antioxidant and ACE inhibitory activities.

[0086] 1.10 Summary

[0087] Peptides prepared from cooked donkey meat exhibit ACE inhibitory and antioxidant activities, but the effects varied under different cooking conditions. The ACE inhibitory activity of donkey meat peptides followed an increase-decrease-increase trend, with the highest ACE inhibitory activity observed in the 65°C / 60 min, 75°C / 30 min, 95°C / 75 min, 75°C / 45 min, and 75°C / 90 min treatments. The ABTS free radical scavenging rate and reducing power generally showed a decreasing then increasing trend, while the hydroxyl free radical scavenging rate exhibited the opposite trend, initially increasing then decreasing. The DPPH free radical scavenging rate increased with time, with the exception of the 45°C and 65°C treatments. Normalization of the four antioxidant indices revealed that the antioxidant activity was highest in eleven treatments, including the 55°C / 90 min, 95°C / 75 min, and 75°C / 90 min treatments. Overall, the 95°C / 75 min treatment group exhibited the highest ACE inhibitory and antioxidant activities.

[0088] Example 2

[0089] 2.1 Structural identification and screening of active peptides

[0090] The present invention has selected six peptide segments with ACE inhibitory activity and antioxidant activity. The length of these six peptide segments is between 3-13 and the molecular weight is between 404.21-1686.85. PeptideRanker is an online website that uses machine learning algorithms, such as support vector machines (SVM), to score the biological activity of peptides based on features such as amino acid composition, length and hydrophobicity. The higher the score, the stronger the potential biological activity of the peptide. The Peptide Ranker scores of these six peptide segments are all greater than 0.90, indicating that these peptides may have potential biological activity. There are no relevant reports by searching the BIOPEP database and literature, indicating that new active peptides have been obtained. The present invention has selected three peptide segments with stability as shown in Table 5.

[0091] Table 5 Active peptides from donkey meat

[0092]

[0093] 2.2 Prediction of physicochemical properties of active peptides

[0094] The physicochemical properties of the peptides are shown in Table 6. All three peptides exhibit hydrophobicity, which may be related to the proportion of hydrophobic amino acids contained in the peptides and the terminal amino acid residues. Studies have shown that ADMET prediction (Table 6) shows that the BBB rates of IGF, IHWPTGF, and AAPF are ≥0.1, showing very high blood-brain barrier rates, which may also be related to the hydrophobicity of the molecules. The HIA rates of peptides IGF and ALF are greater than 0.3, indicating good human intestinal absorption. The allergenicity of low-molecular-weight peptides is lower than that of the proteins they produce, and the SVM scores are less than zero, indicating that all three peptides are non-toxic.

[0095] Table 6 Prediction of physicochemical properties of donkey meat active peptides

[0096]

[0097] 2.3 Analysis of the docking site between the active peptide and the ACE molecule

[0098] Molecular docking is an effective method for exploring the interaction mechanism between ACE and inhibitory peptides. Computer-assisted prediction of the mode of action, binding capacity, and affinity of ACE inhibitory peptides with ACE can be used. The binding energies and interaction sites between the peptides and ACE are shown in Table 7. All three peptides bind to ACE with low binding energies, with IHWPTGF having the lowest binding energy (-115.33 kcal / mol) and AAPF having the highest binding energy (-72.45 kcal / mol). Lower binding energies indicate more stable binding to ACE and stronger inhibitory activity. Therefore, based on the binding energies, IHWPTGF may possess potent ACE inhibitory activity.

[0099] Table 7 Binding energy and chemical interactions of peptides docked with ACE molecules

[0100]

[0101] Both IHWPTGFIGF and AAPF can be combined with Zn 2+ The amino acids in the active center interact with each other to reduce ACE activity in a competitive inhibitory manner. The results of the present invention show that Thr of IHWPTGF forms two hydrogen bonds with Glu411 and His387, and IHWPTGF also has a hydrophobic interaction with His387. Gly of IGF forms three hydrogen bonds with Glu411; Ile forms one hydrogen bond with His383 and His387 respectively, and Ile also has charge attraction and hydrophobic interaction with Glu411 and His383 respectively. Ala (A1) in AAPF forms one hydrogen bond with His383 and a charge attraction with Glu411; Ala (A2) forms one hydrogen bond with His387. Zn 2+ Zn plays a key role in maintaining ACE activity. 2+Glu411, His387 and His383 in the active center bind to zinc ions to form a tetrahedral coordination, and the amino acids in the active peptide inhibit ACE activity through interactions with the tetrahedral coordination of zinc ions.

[0102] like Figure 7 As shown in Figure A, the Gly residue of IHWPTGF interacts with the S1 pocket and forms two hydrogen bonds with amino acid residues Glu384 and Tyr523 in ACE. IHWPTGF also has a hydrophobic interaction with Ala354. Phe forms two hydrogen bonds with Tyr520 and His353 in the S2 pocket. Phe also forms a charge attraction with Lys511 in the S2 pocket, and there is also a hydrophobic interaction between IHWPTGF and His354. Ala354 and Lys511 are reported to be important residues in ACE and interact with the antihypertensive drug lisinopril. Many peptides with strong ACE inhibitory activity bind to Ala354 or Lys511. Therefore, binding to the active site at Ala354 and Lys511 may partially explain the strong ACE inhibitory activity of IHWPTGF.

[0103] like Figure 7 As shown in B, Ile at the amino terminus of IGF forms two hydrogen bonds with amino acids in the S1 pocket. In addition to the hydrogen bond, the interaction with Glu384 also forms a charge attraction. At the same time, there is a hydrophobic interaction between Ile and Ala354. In addition, Ile forms a hydrophobic interaction with His353 and His513 in the S2 pocket. Ala (A1) in AAPF forms two hydrogen bonds with Glu384 and Tyr523 in the S1 pocket, and also forms a charge attraction with Glu384 ( Figure 7 C), the study showed that molecular interactions through hydrogen bonding and attractive charges contribute to the stability of the formed complex. Peptides IHWPTGF, IGF, and AAPF formed 12, 10, and 7 hydrogen bonds with ACE, 23 and 3 attractive charges, and 9, 5, and 1 hydrophobic interactions, respectively. In general, the donkey meat ACE inhibitory peptides have strong affinity with the S1 pocket and Zn 2+ The main interaction between the residues is hydrogen bonding, and the main hydrophobic interaction with pocket S2 is generated (Pi-Pi and Pi-alkyl interactions). It is speculated that the donkey meat ACE inhibitory peptide interacts with the active site of ACE through hydrogen bonding and some short-range The molecular interactions between the active peptides and the MPO molecules are the result of attractive charges, which stabilizes the complex. The C-termini of IHWPTGF, ALGF, and IGF all have Phe, which may be one of the reasons why the peptides have a good affinity with ACE. 2.4 Analysis of the docking sites between the active peptides and the MPO molecules

[0104] MPO is the main enzyme involved in the production in vivo and is a heme peroxidase in neutrophils and monocytes. In the presence of H2O2 and halides, MPO produces ROS, leading to cell and tissue damage. In theory, active peptides can inhibit enzyme activity by occupying the active site of the enzyme or by blocking the contact between the substrate and the active site cavity. The molecular docking results show that, as shown in Table 8, IHWPTGF, IGF, and AAPF are stably and well docked to the catalytic active site of MPO, indicating that all three peptides can interact with the heme group. IHWPTGF has the strongest affinity. IHWPTGF forms 8 hydrogen bonds, 4 charge attractions, and 7 hydrophobic interactions with MPO. Figure 8 A, IHWPTGF forms three hydrophobic interactions and one charge attraction with His336 and Glu242 on the C chain. His in IHWPTG also forms a hydrogen bond with His336. Furthermore, IHWPTGF and AAPF bind to the cleft of the active site through electrostatic interactions with Asp94 and Asp98 on the MPOA chain. The His in the IHWPTGF peptide contains an imidazole functional group. The imidazole group and aromatic ring interact with the peptide through electron transfer / proton transport, providing free radical scavenging capabilities, a characteristic of many food-derived antioxidant peptides.

[0105] Table 8 Binding energy and chemical interactions between peptides and MPO molecules

[0106]

[0107] ALF forms 7 hydrogen bonds, 3 charge attractions and 2 hydrophobic interactions with MPO. Ala of ALF forms 1 hydrogen bond with Arg239 of C chain and His95 of A chain respectively. In addition, Leu also forms 1 hydrogen bond with His336 of C chain ( Figure 8 B) IGF forms 6 hydrogen bonds, 3 charge attractions and 5 hydrophobic interactions with MPO. Figure 8 As shown in Figure C, IGF forms a hydrophobic interaction with the key amino acid His336; the pi-pi interaction promotes charge transfer between the ligand and the receptor. In addition, there are several other favorable interactions (hydrogen bonds, salt bridges, and attractive charges) between non-catalytic residues and IGF.

[0108] The results showed that IHWPTGF, ALF, and IGF can all form hydrogen bonds or hydrophobic interactions with His336, which is consistent with the molecular docking results of tuna protein antioxidant peptides and MPO. The MPO docking postures of these three peptides all blocked the entrance to the MPO active cavity, confirming that they are effective antioxidants. Among the above peptides, IHWPTGF has the strongest affinity for MPO, and only IHWPTGF forms three forces with MPO: hydrogen bonds, charge attraction, and hydrophobic interactions. It is speculated that IHWPTGF has the strongest antioxidant activity. In summary, IHWPTGF, AAPF, and IGF show good binding affinity with key amino acid residues of MPO. The binding sites of these peptides can prevent substrates (such as H2O2) from entering the core of MPO, thereby exerting an antioxidant effect.

[0109] 2.5 Analysis of the stability of active peptides

[0110] In silico hydrolysis was used to analyze the stability of peptides identified by LC-MS / MS.

[0111] Table 9 lists the peptides and possible fragments released after gastrointestinal enzymatic hydrolysis. Computer-simulated hydrolysis results indicate that under ideal computer-simulated conditions, the peptide bonds of IHWPTGF, AAPF, and IGF remain intact. Some researchers believe that the structure of a peptide influences its resistance to digestive enzymes in the body, and that the presence of proline can increase its resistance to hydrolysis by digestive enzymes. The presence of proline residues in the peptides IHWPTGF and AAPF may allow them to survive longer in the digestive system and increase their potential for biological activity in vivo.

[0112] Table 9 Peptides and fragments that may be released after exposure to gastrointestinal enzymatic degradation

[0113]

[0114] 2.6 Peptide activity verification

[0115] To verify our prediction, IHWPTGF, AAPF, and IGF were synthesized and their ACE inhibitory and antioxidant activities were evaluated. As shown in Table 10, the ACE inhibitory activity of IHWPTGF>IGF>AAPF (P<0.05), IC 50 The values ​​were 0.32, 0.53, and 1.16 mg / mL respectively; IHWPTGFABTS had the strongest free radical scavenging ability, followed by IGF, and AAPF had the weakest ability. 50 The values ​​were 2.23 mg / mL, 3.23 mg / mL, and 3.73 mg / mL, respectively; the DPPH free radical scavenging ability was IHWPTGF>AAPF>IGF (P<0.05), IC 50The values ​​were 0.81, 1.75, and 2.04 mg / mL, respectively; the hydroxyl radical scavenging ability was IHWPTGF>AAPF>IGF (P<0.05), IC 50 The values ​​were 0.64, 0.94, and 0.97 mg / mL, respectively; when the concentration was 2 mg / mL, the reducing abilities of IHWPTGF, AAPF, and IGF were 26.62, 24.57, and 22.47, respectively. The reducing abilities of the synthetic peptides were IHWPTGF>AAPF>IGF (P<0.05).

[0116] The ACE inhibitory activity of IHWPTGF is higher than that of many peptides with ACE inhibitory activity, such as YRKER (IC 50 =3.3 mg / mL), LHLPLP (IC 50 =13.6mg / mL). The peptide IHWPTGF has a representative structural model, including five hydrophobic amino acids: Ile, Trp, Pro, Gly, and Phe, with a positively charged His in the middle, which may facilitate ACE binding. IHWPTGF exhibits superior free radical scavenging and iron ion reduction abilities compared to the other two peptides, consistent with the molecular docking results. This is likely due to the formation of a greater number of molecular interactions between IHWPTGF and the key active site of ACE. Furthermore, the His in the peptide contains an imidazole functional group, which enhances free radical scavenging through electron transfer and proton donation. Therefore, IHWPTGF also exhibits excellent antioxidant capacity. In summary, IHWPTGF exhibits strong ACE inhibitory and antioxidant activities and has broad application prospects in nutrition and pharmaceuticals.

[0117] Table 10 Verification of ACE inhibitory activity and antioxidant activity of synthetic peptides

[0118]

[0119] 2.7 Summary

[0120] Based on the characteristics of peptides reported to exhibit ACE inhibitory and antioxidant activities, three new peptides were identified. All peptides had PeptideRanker scores greater than 0.90, indicating potential bioactivity. Physicochemical properties and ADMET prediction revealed that all three peptides exhibited hydrophobicity, with IGF, IHWPTGF, and AAPF exhibiting very high blood-brain barrier clearance. The peptides IGF and ALGF exhibited good intestinal absorption in humans, and all three peptides were non-toxic. The three peptides, IHWPTGF, AAPF, and IGF, all showed good affinity for ACE and MPO and demonstrated stable functional activity. IHWPTGF exhibited the strongest ACE inhibitory and antioxidant activity, consistent with molecular docking results and activity validation of the synthetic peptides.

[0121] Example 3

[0122] 3.1 Peptide preparation

[0123] The donkey meat polypeptide was obtained from Example 1 (the specific cooking conditions were as follows: the cooking bag containing the meat was placed in a water bath and heated at 95° C. for 75 minutes. After heating, the cooking bag was immersed in ice water for 10 minutes to stop the thermal reaction).

[0124] 3.2 Preparation process of peptide health products Figure 13 shown.

[0125] 3.3 Operation points

[0126] (1) Filtration, deodorization, and sterilization: After the enzymatic hydrolysis is completed, the mixed solution is centrifuged (4°C, 20 min), the supernatant is filtered with filter paper, the filtrate is passed through a 0.22 μm membrane, the filtrate is mixed with the deodorizer, and sterilized with high-pressure steam for 30 min at the corresponding temperature and time for deodorization treatment.

[0127] (2) Preparation and mixing: Prepare donkey meat polypeptide, citric acid, xylitol and lemon essence in proportion, and then mix the sample with a multi-functional grinder.

[0128] (3) Packaging: Packed in 10g / bag.

[0129] 3.4 Deodorant Screening

[0130] During the enzymatic hydrolysis process, lipid oxidation and protein degradation cause meat peptides to acquire a fishy odor, which is typically removed using a deodorant. The primary method for screening deodorants is a scoring method. Ten professionally trained sensory evaluators were randomly selected to rate the fishy odor of donkey meat peptide solutions. The control group consisted of deionized water, with scores of 5, 4, 3, 2, 1, and 0 corresponding to heavy, heavy, moderate, light, light, and no odor, respectively. The deodorization conditions are shown in Table 11. After deodorization, the mixed solution was centrifuged (4°C, 20 min), and the supernatant was filtered through filter paper.

[0131] Table 11 Deodorizing agents and deodorizing conditions

[0132]

[0133] 10g of donkey meat peptide solid health supplement was diluted with 100mL of water (40°C). Ten professionally trained sensory evaluators then rated the supplement on taste, flavor, and texture. The participants abstained from stimulating foods before the evaluation. The evaluation criteria are shown in Table 12.

[0134] Table 12 Sensory evaluation standards for donkey meat polypeptide solid health products

[0135]

[0136] The deodorization effect of solid health products is shown in Table 14. The lower the sensory score, the better the deodorization effect. Although different deodorization conditions had different effects, the deodorization effect was improved to a certain extent compared to the blank control group. The sensory scores ranked from high to low as follows: blank > activated carbon > active dry yeast > β-cyclodextrin > active dry yeast + β-cyclodextrin. Activated carbon was not very effective in deodorizing, while active dry yeast and β-cyclodextrin were more effective but had a slight fishy smell. Active dry yeast + β-cyclodextrin was the most effective in deodorizing, and yeast deodorization also produced a unique aroma, enhancing the flavor. Therefore, the active dry yeast + β-cyclodextrin combination was selected for deodorization. The active dry yeast dosage was 0.2%, the deodorization temperature was 35°C, and the deodorization time was 60 minutes. The β-cyclodextrin dosage was 0.2%, the deodorization temperature was 60 minutes, and the deodorization time was 40 minutes.

[0137] Table 14 Sensory evaluation of the deodorization effect of donkey meat polypeptide solid health product

[0138]

[0139] 3.5 Single-factor experimental design

[0140] Sensory scores were used as indicators to investigate the effects of different addition amounts of donkey meat peptide, citric acid, xylitol, and lemon essence on the sensory quality of donkey meat peptide solid health products. The single factor conditions are shown in Table 13.

[0141] Table 13 Single factor experimental conditions of donkey meat polypeptide solid health product

[0142]

[0143] like Figure 9 As shown in the figure, the sensory score of the solid health product shows a trend of first increasing and then decreasing with the increase of the donkey meat polypeptide dosage. The highest point is 2.50g, with the highest value of (85.7±0.67). At this time, the solid health product has no lumps and no odor. The liquid after reconstitution is uniform light yellow and has a good taste. When the amount of donkey meat polypeptide added exceeds 2.50g, the sensory quality decreases. Too much addition will produce an unpleasant odor, and will cause the tissue state to be turbid or even produce precipitation, affecting the sensory quality of the health product. When the amount of donkey meat polypeptide added is less than 2.50g, the health product lacks a unique flavor and does not have a mellow overall taste. Therefore, three levels of donkey meat polypeptide addition of 2.00g, 2.50g, and 3.00g were selected for orthogonal experiments.

[0144] like Figure 10As shown in the figure, when the amount of citric acid added is 0.05g-0.20g, the sensory quality of the solid health product increases with the increase of the amount of citric acid added. When the highest point is 0.20g, the sensory score value reaches (87.20±0.79). At this time, the taste of the solid health product is suitable, with moderate sweet and sour taste, no peculiar smell, and a good tissue state. When the amount of citric acid added exceeds 0.20g, the sensory quality decreases. Citric acid can cover the bitterness to a certain extent, but too much addition leads to enhanced sourness, an uncoordinated taste, and cannot cater to the taste of the public. When the amount of citric acid added is less than 0.20g, the mouthfeel after brewing is lighter and the overall taste is insufficient. Therefore, the orthogonal experiment is carried out by selecting three levels of citric acid addition: 0.15g, 0.20g, and 0.25g.

[0145] like Figure 11 As shown, when the amount of xylitol added is 2.00g-8.00g, the sensory quality of the solid health product shows an upward trend as the amount of addition increases. When the amount of addition is 8.0g, the sensory score is the highest (85.7±0.82). The solid health product at this time has no lumps, tastes sweet and sour after mixing, has a uniform and bright color, and a mellow taste. When the amount of xylitol added exceeds 8.00g, the sensory quality decreases, and the amount of addition causes the health product to be too sweet. When the amount of addition is less than 8.00g, the overall taste of the solid health product is lighter and cannot meet the needs of the public. Therefore, xylitol addition amounts of 6.00g, 8.00g, and 10.00g are selected for orthogonal experiments.

[0146] like Figure 12 As shown in the figure, the sensory quality of solid health products shows a trend of first increasing and then decreasing with the increase of lemon flavor. When the addition amount is 0.10g-0.20g, the sensory quality increases, and the highest point is 0.20g, when the sensory score reaches (81.8±1.03); thereafter, the sensory quality decreases with the increase of the addition amount of lemon flavor. Adding excessive lemon flavor will lead to a decrease in the solubility of solid health products, making the color distribution of the health products uneven, and causing its tissue state to be turbid or even precipitated. Too much lemon flavor will also produce a sour taste, affecting the taste of the health products, thereby affecting the sensory quality of the solid health products. Therefore, 0.15g, 0.20g, and 0.25g of lemon flavor were selected for orthogonal experiments.

[0147] 3.6 Orthogonal experiment

[0148] Using sensory scores as the evaluation criteria, on the basis of single factor experiments, the addition amounts of donkey meat polypeptide (A), citric acid (B), xylitol (C) and fruit essence (D) were selected as the investigation factors to carry out L9(3 4 ) Orthogonal experiments were conducted to optimize the formula of donkey meat polypeptide solid health supplements. The coding table for each factor level is shown in Table 15.

[0149] Table 15 Orthogonal experimental design table (g)

[0150]

[0151] Table 16 Orthogonal experiment results of donkey meat polypeptide solid health products

[0152]

[0153]

[0154] Table 17 Test of inter-subject effects

[0155]

[0156] Note: R 2 =0.965(Adjusted R 2 =0.949)

[0157] In the orthogonal experiment, sample 6 (i.e., A2B3C1D2) performed best with a score of 88.67, making it the optimal formula for the orthogonal experiment (Table 16). Range analysis revealed that the factors influencing the sensory evaluation of donkey meat polypeptide solid health products were, in order: the amount of donkey meat polypeptide (A), citric acid (B), lemon flavor (D), and xylitol (C). A comparative analysis of sensory scores showed that A2B3C1D2 was the optimal formula for the donkey meat polypeptide solid health product, consistent with the results of sample 6. Therefore, the optimal formula combination was A2B3C1D2, consisting of 2.50g donkey meat polypeptide, 0.25g citric acid, 6.00g xylitol, and 0.20g lemon flavor.

[0158] 3.7 Finished Product Quality Analysis The indicators of the polypeptide solid health product were measured, and the results are shown in Table 18. The protein content of the donkey meat polypeptide solid health product is higher than the 0.5g / 100mL specified in "GB / T 29602-2013 Solid Beverages", and the hygiene and sensory indicators are excellent. It also has good ACE inhibitory activity and antioxidant activity.

[0159] Table 18 Donkey meat polypeptide solid health product index test results

[0160]

[0161] 3.8 Summary

[0162] The present invention adopts four deodorization methods, namely activated carbon, beta-cyclodextrin, active dry yeast, and active dry yeast + beta-cyclodextrin, to deodorize donkey meat polypeptide. The results show that the deodorization effect of active dry yeast + beta-cyclodextrin is the best. The deodorization dosage is active dry yeast (0.2%) + beta-cyclodextrin (2.0%), the temperature is active dry yeast (35°C) + beta-cyclodextrin (60°C), and the time is active dry yeast (60 minutes) + beta-cyclodextrin (40 minutes). The deodorized donkey meat polypeptide powder is used as raw material, and the addition amount of donkey meat polypeptide, citric acid, xylitol, and lemon essence is studied to prepare a donkey meat polypeptide solid health product. Based on the single-factor experiments, orthogonal experiments were conducted to determine the optimal solid health product formula: 2.50g (28%) of donkey meat peptide, 0.25g (3%) of citric acid, 6.00g (67%) of xylitol, and 0.20g (2%) of lemon essence. The resulting solid health product has a moderate sweet-sour flavor, a uniform texture, a translucent color, and a unique aroma. Quality testing of the donkey meat peptide solid health product revealed that it exhibited excellent ACE inhibitory and antioxidant activity, suggesting broad application prospects and potential for subsequent product development.

[0163] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and embodiments. They can be applied to a variety of fields suitable for the embodiments of the present invention. Those skilled in the art will readily realize further modifications. Therefore, without departing from the general concept defined by the claims and their equivalents, the embodiments of the present invention are not limited to the specific details and illustrations shown and described herein.

Claims

1. A bifunctional peptide segment with ACE inhibitory activity and antioxidant activity, characterized in that: The amino acid sequence of the bifunctional peptide segment is IHWPTGF or IGF.

2. A method for preparing a donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity, characterized in that: The steps include: 1) Heat the donkey meat at 45-95°C for 30-90 minutes, then stop the thermal reaction. 2) The heated donkey meat is sequentially subjected to in vitro simulated gastric biomimetic enzymolysis and in vitro simulated intestinal biomimetic enzymolysis to obtain a donkey meat polypeptide solution containing a bifunctional peptide segment with ACE inhibitory activity and antioxidant activity, wherein the amino acid sequence of the bifunctional peptide segment with ACE inhibitory activity and antioxidant activity is IHWPTGF or IGF.

3. The method for preparing a donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity as claimed in claim 2, wherein: In step 1), cut the donkey meat into blocks of 3×3×2 cm.

4. The method for preparing a donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity as claimed in claim 3, wherein: In step 1), the donkey meat raw material is heated at 95°C for 75 minutes.

5. The method for preparing a donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity as claimed in claim 2, wherein: In step 2), the in vitro simulated gastric biomimetic enzymatic hydrolysis method includes: taking heated donkey meat, mincing it and adding it to water, with the mass volume ratio of donkey meat to water being 1:4, after homogenization, adjusting the pH value to 2.0 ± 0.1, adding 40,000 U / g pepsin accounting for 1% of the volume of the reaction system, and enzymatically hydrolyzing it at 37°C under a constant temperature and vibration environment for 2 hours.

6. The method for preparing a donkey meat polypeptide comprising a bifunctional peptide segment having ACE inhibitory activity and antioxidant activity as claimed in claim 2, wherein: In step 2), the method for in vitro simulated intestinal biomimetic enzymatic hydrolysis includes: after performing the in vitro simulated gastric biomimetic enzymatic hydrolysis, adjusting the pH value of the enzymatic hydrolysis solution to 7.5 ± 0.1, adding 40,000 U / g trypsin solution accounting for 1% of the volume of the reaction system, and simulating in vitro intestinal fluid enzymatic hydrolysis for 2 hours at 37°C under a constant temperature and vibration environment.

7. A method for preparing a donkey meat polypeptide health product comprising the bifunctional peptide segment according to claim 1, characterized in that: The steps include: 1) Heat the donkey meat at 45-95°C for 30-90 minutes, then stop the thermal reaction. 2) subjecting the heated donkey meat to in vitro simulated gastric biomimetic enzymatic hydrolysis and in vitro simulated intestinal biomimetic enzymatic hydrolysis, thereby obtaining a donkey meat polypeptide solution containing bifunctional peptides having ACE inhibitory activity and antioxidant activity; 3) After the enzymatic hydrolysis is completed, the donkey meat polypeptide solution is filtered to obtain a filtrate, the filtrate is mixed with a deodorizing agent to perform a deodorizing treatment, and then sterilized and dried to obtain a donkey meat polypeptide powder, wherein the deodorizing treatment is performed by any one of the following methods: Active dry yeast with a concentration of 0.2% was used to remove the fishy smell at 35°C for 60 minutes; Use 2.0% β-cyclodextrin to remove the fishy smell at 60°C for 40 minutes; or First, 0.2% active dry yeast was used to remove the fishy smell at 35°C for 60 minutes, and then 2.0% β-cyclodextrin was used to remove the fishy smell at 60°C for 40 minutes. 4) The donkey meat polypeptide powder is formulated to obtain a donkey meat polypeptide health product, which is used for non-pharmaceutical purposes.

8. The method for preparing the donkey meat polypeptide health product according to claim 7, wherein: In step 4), citric acid, xylitol and lemon essence are prepared, and the mass ratio of the donkey meat polypeptide powder to citric acid, xylitol and lemon essence is: 20-30:1.5-2.5:60-100:1.5-2.

5.

9. The method for preparing the donkey meat polypeptide health product according to claim 8, wherein: The mass ratio of the donkey meat polypeptide powder to citric acid, xylitol and lemon essence is 25:2.5:60:

2.

10. A donkey meat polypeptide or donkey meat polypeptide health product comprising the bifunctional peptide segment according to claim 1, characterized in that: The donkey meat polypeptide is prepared by the method according to any one of claims 3 to 6, the donkey meat polypeptide health product is prepared by the method according to any one of claims 7 to 9, and the donkey meat polypeptide health product is used for non-pharmaceutical purposes.

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