Preparation method of bovine myocardial antioxidant peptide and compound particles thereof

Abstract

The invention relates to the technical field of biological enzymolysis, and provides a preparation method of bovine myocardial antioxidant peptide and composite particles thereof. The bovine myocardial antioxidant peptide is prepared mainly through a two-step enzymolysis method, and bovine myocardial peptide is encapsulated by adopting a protein-polysaccharide composite embedding method so as to improve the stability of the bovine myocardial peptide. The bovine myocardial antioxidant peptide is prepared by compounding two proteases, so that the production efficiency and the product quality are improved, and meanwhile, the DPPH free radical scavenging rate of the bovine myocardial peptide is remarkably increased. Soybean protein isolate and sodium alginate are selected as packaging materials to package the bovine myocardial peptide, so that oxidative decomposition of the bovine myocardial peptide due to influence of illumination, oxygen, metal ions and the like or pH, enzyme and other factors in an organism in the processing, storage and transportation processes is inhibited, and the bioavailability of the bovine myocardial peptide is further improved. The obtained bovine myocardial peptide is green, safe and efficient, and the prepared composite particles effectively improve the stability of the bovine myocardial antioxidant peptide and have wide application prospects in the fields of food, medicine, cosmetics and the like.

Description

Technical Field

[0001] This invention mainly relates to the field of bioenzymatic hydrolysis technology, specifically a two-step enzymatic hydrolysis method for efficiently preparing bovine myocardial antioxidant peptides, and employing a protein-polysaccharide composite encapsulation method to encapsulate the extracted bovine myocardial peptides to improve their stability. Background Technology

[0002] In recent years, my country's animal husbandry industry has shown a booming development trend, but it also faces a key problem: the processing and utilization of by-products obtained from the slaughter and processing of mammals, such as bones, blood, heart, liver, and stomach, is relatively low. In traditional market transactions, animal by-products are often sold at a lower price than other muscle parts, or used as raw materials to produce low-value-added products such as animal feed and pet food, resulting in a serious waste of resources to some extent. Meanwhile, with the continuous upgrading of market demand and the transformation of social concepts regarding resource utilization, deeply exploring the potential value of animal by-products and utilizing them for high-value purposes can not only reduce resource waste and achieve optimal resource allocation, but also open up new economic growth areas for animal husbandry, powerfully promoting the entire industry towards a more efficient and sustainable direction.

[0003] Current research indicates that bioactive peptides possess the ability to precisely regulate physiological homeostasis, enhance immune system efficacy, and efficiently scavenge free radicals within the body. Antioxidant peptides, as an important branch of bioactive peptides, can effectively eliminate excess free radicals, inhibit lipid peroxidation, and slow down cellular oxidative damage, thus possessing significant health benefits. Compared to commonly available chemically synthesized antioxidants, natural antioxidant peptides extracted from animals or plants not only effectively maintain the dynamic balance of the body's oxidative system, creating a relatively stable internal environment, but are also safer, more efficient, and more easily absorbed by the body.

[0004] Bovine myocardium, a high-quality animal protein with great development potential, is rich in protein, amino acids, vitamins, and other nutrients essential for the human body, making it a superior raw material for preparing bioactive peptides. However, most commercially available myocardial peptide products are prepared using traditional methods such as repeated freeze-thaw water extraction or single enzymatic hydrolysis. The resulting bioactive peptides suffer from drawbacks such as low purity, large molecular weight, and low extraction rate, limiting their application and promotion in high-end fields. Therefore, to improve the production efficiency and product quality in the preparation of bovine myocardial antioxidant peptides, this invention proposes a highly efficient, green, and economical preparation method. This method not only provides a new approach to the extraction and preparation of antioxidant peptides but also promotes the sustainable and healthy development of related industries and facilitates the efficient conversion and utilization of mammalian by-products.

[0005] However, natural antioxidant peptides are chemically unstable and extremely susceptible to oxidative decomposition during processing, storage, and transportation due to factors such as light, moisture, oxygen, and metal ions. Furthermore, they are easily hydrolyzed in vivo by factors such as temperature, pH, and enzymes. Encapsulating bovine cardiac muscle antioxidant peptides using soy protein isolate and sodium alginate as packaging materials not only effectively inhibits the breakdown of bovine cardiac muscle peptides into amino acids in vivo, allowing for greater absorption by the body in the form of small peptide molecules, but also effectively solves the problem of the chemical instability of bovine cardiac muscle peptides, expanding their application in antioxidant foods, pharmaceuticals, and cosmetics. Summary of the Invention

[0006] To address the key technical challenges in the preparation of antioxidant peptides using enzymatic hydrolysis, this invention provides a method for preparing bovine myocardial antioxidant peptides and their composite microparticles. The aim is to solve problems such as resource waste in bovine myocardium, low additional utilization rate, and the inherent chemical instability of small molecule peptides. This significantly improves the production efficiency, product quality, and bioavailability of bovine myocardial antioxidant peptides, while also expanding the application scope of natural antioxidant peptides in food, medicine, and cosmetics.

[0007] To address the aforementioned technical problems, the present invention provides the following technical solution:

[0008] Fresh bovine heart muscle was cleaned, and fat and connective tissue were removed. The homogenate was prepared at a ratio of bovine heart muscle to deionized water of 1:4 (g / mL). The homogenate was sterilized in a boiling water bath for 15 min and then rapidly cooled to room temperature to obtain the raw material solution. The pH was adjusted to 8.0, and alkaline protease was added for the first enzymatic hydrolysis (hydrolysis temperature: 44.70 ℃, time: 2.06 h, enzyme dosage: 0.47%). Hydrolysate I was rapidly cooled to room temperature, and its pH was adjusted to 7.41. Trypsin was added for the second enzymatic hydrolysis (hydrolysis conditions: temperature: 37.0 ℃, time: 3.5 h, enzyme dosage: 0.05%). Hydrolysate II was boiled in a boiling water bath for 25 min to inactivate the enzyme. After cooling to room temperature, it was centrifuged at 4000 r / min for 10 min, and the supernatant was collected.

[0009] The supernatant obtained was freeze-dried to obtain bovine myocardial antioxidant peptide powder. The antioxidant activity of the obtained bovine myocardial antioxidant peptide was determined, and its DPPH free radical scavenging rate reached 85.43%.

[0010] 0.2 g of soy protein isolate and bovine myocardial antioxidant peptide were dissolved in 10 mL of deionized water and stirred continuously at room temperature for 3.0 h. The pH of the solution was then adjusted to 4.0. 0.1 g of sodium alginate was dissolved in 10 mL of deionized water until the solution was completely transparent. The soy protein isolate-bovine myocardial peptide solution was slowly added to the sodium alginate solution, and the pH of the mixture was adjusted to 3.5. The mixture was stirred continuously at room temperature for 1.0 h. After centrifugation, the supernatant was allowed to stand at 4 ℃ for 24 h and then freeze-dried to obtain composite microparticles with soy protein isolate and sodium alginate as carriers.

[0011] The encapsulation efficiency, antioxidant activity, and in vitro gastrointestinal digestion simulation of the obtained composite microparticles were measured. The encapsulation efficiency was 66.65%, the DPPH free radical scavenging rate was 59.59%, the absorbance value of ferric ion reducing power was 0.181, and the peptide retention rate after gastrointestinal digestion was 25.31%. Compared with the control group of antioxidant peptides, the retention rate of small molecule peptides after gastrointestinal digestion was significantly improved, laying the foundation for the widespread application of bovine myocardial antioxidant peptides in many fields such as medicine, food, and skin care products. Attached Figure Description

[0012] Appendix Figure 1 The figure shows the effect of alkaline protease hydrolysis time on bovine myocardial antioxidant peptides obtained in Example 2 of the present invention.

[0013] Appendix Figure 2 The graph shows the effect of alkaline protease hydrolysis temperature on bovine myocardial antioxidant peptides obtained in Example 3 of this invention.

[0014] Appendix Figure 3 The graph shows the effect of the amount of alkaline protease added on the antioxidant peptides of bovine myocardium obtained in Example 4 of this invention.

[0015] Appendix Figure 4 The figure shows the effect of trypsin hydrolysis pH on bovine myocardial antioxidant peptides obtained in Example 5 of the present invention.

[0016] Appendix Figure 5 The figure shows the response surface methodology results obtained after optimizing the preparation process of bovine myocardial antioxidant peptides in Example 6 of this invention.

[0017] Appendix Figure 6 The graph shows the encapsulation efficiency measurement results of the composite microparticles obtained in Example 8 of this invention.

[0018] Appendix Figure 7 The graph shows the DPPH free radical scavenging rate of the composite microparticles obtained in Example 8 of this invention.

[0019] Appendix Figure 8 The graph shows the results of the iron ion reducing power measurement of the composite microparticles obtained in Example 8 of the present invention.

[0020] Appendix Figure 9 This is a graph showing the peptide retention rate after in vitro gastrointestinal digestion simulation of the composite microparticles obtained in Example 8 of the present invention.

[0021] Appendix Figure 10 The image shows the bovine myocardial peptides and composite microparticles obtained in this invention. Detailed Implementation

[0022] Example 1

[0023] (1) Take 8.0 g of bovine myocardium, clean it, remove connective tissue and fat, homogenize it according to the ratio of bovine myocardium:deionized water = 1:4 (g / mL), sterilize it in boiling water bath for 15 min, and then quickly cool it to room temperature. Add 0.70% of the weight of bovine myocardium alkaline protease (adjust pH to 11.0, temperature 50.0 ℃), papain (adjust pH to 6.0, temperature 55.0 ℃), flavor protease (adjust pH to 7.0, temperature 52.0 ℃) and pepsin (adjust pH to 3.0, temperature 42.0 ℃) to hydrolyze it for 2.5 h to obtain hydrolysate I;

[0024] (2) After cooling the enzymatic hydrolysate I to room temperature, adjust its pH to 7.8, add trypsin for enzymatic hydrolysis. The enzymatic hydrolysis conditions are: temperature 37.0 ℃, time 3.5 h, and enzyme addition amount of 0.09% of bovine myocardium weight, to obtain enzymatic hydrolysate II;

[0025] (3) After inactivating the enzyme in the enzymatic hydrolysate II by boiling water bath for 25 min, quickly cool it to room temperature, centrifuge at 4000 r / min for 10 min, and take the supernatant.

[0026] (4) The peptide content, DPPH free radical scavenging rate and hydroxyl free radical scavenging rate of the supernatant of different groups of samples were determined.

[0027] (5) Determination of peptide content

[0028] Preparation of standard curve: Using bovine serum albumin as the standard, a 10 mg / ml standard solution was prepared with deionized water. Six test tubes were filled with 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution, respectively. Deionized water was added to a final volume of 1.0 mL, followed by 4.0 mL of biuret reagent. The mixture was mixed and allowed to stand in the dark for 30 min. The supernatant was then measured at a wavelength of 540 nm. The first group without protein solution was used as a blank control. Protein content was plotted on the x-axis, and absorbance was plotted on the y-axis.

[0029] Sample determination: Place 1.75 mL of sample solution in a test tube, add 0.75 mL of 5% trichloroacetic acid, mix well, let stand for 30 min, centrifuge at 6000 r / min for 10 min, transfer the supernatant to a 25 mL volumetric flask, and dilute to the mark with 5% trichloroacetic acid. Mix 1.0 mL of the mixture with 4.0 mL of biuret reagent, let stand in the dark for 30 min, and measure the absorbance of the supernatant at 540 nm. Zero the sample using a 1:4 (v / v) mixture of deionized water and biuret reagent, and then calculate the polypeptide content in the enzymatic hydrolysate.

[0030] (6) Determination of DPPH free radical scavenging rate

[0031] Preparation of test sample solution: Add 4.0 mL of the test solution and 0.2 mmol / L DPPH-95% ethanol mixture and mix well. After standing in the dark at room temperature for 30 min, centrifuge at 6500 r / min for 10 min and take the supernatant. Detect its absorbance value at a wavelength of 517 nm.

[0032] Preparation of the control group for the test sample: Take 4.0 mL of the sample solution and mix it with 95% ethanol solution. After treatment under the same conditions, measure its absorbance value.

[0033] Preparation of blank control samples: Mix 4.0 mL of 0.2 mmol / L DPPH-95% ethanol solution and deionized water, shake well, treat under the same conditions, and then measure the absorbance. The formula for calculating the DPPH free radical scavenging rate is as follows:

[0034] Equation (1)

[0035] In the formula, A is the absorbance value of the sample solution group to be tested; A1 is the absorbance value of the control group of the sample to be tested; and A0 is the absorbance value of the blank group of the sample to be tested.

[0036] (7) Determination of hydroxyl radical scavenging rate

[0037] Aliquots of 0.5 mL of salicylic acid ethanol solution (10 mM), 0.5 mL of FeSO4 solution (10 mM), and 3.5 mL of deionized water were added to a 0.5 mL sample. The Fenton reaction was initiated by adding 5.0 mL of H2O2 (100 mM) and incubated at 37°C for 1.0 h. Deionized water was used instead of the sample solution and H2O2 as the blank and control groups, respectively. The absorbance of the mixture at 510 nm was then measured. The calculation formula is as follows:

[0038] Equation (2)

[0039] In the formula A s A * and A c These represent the absorbance values ​​of the sample group, control group, and blank group, respectively.

[0040] Example 2

[0041] (1) Take 8.0 g of bovine myocardium, clean it, remove connective tissue and fat, homogenize it according to the ratio of bovine myocardium:deionized water = 1:4 (g / mL), sterilize it in boiling water bath for 15 min and cool it rapidly to room temperature, adjust its pH to 11.0, add alkaline protease for enzymatic hydrolysis, and obtain enzymatic hydrolysate I; the enzymatic hydrolysis conditions are temperature 50.0 ℃, enzyme addition amount of 0.70%, and time of 1.5~3.5 h.

[0042] (2) After cooling the enzymatic hydrolysate I to room temperature, adjust its pH to 7.8, add trypsin for enzymatic hydrolysis to obtain enzymatic hydrolysate II. The enzymatic hydrolysis conditions are: temperature 37.0 ℃, time 3.5 h, and enzyme addition amount of 0.09%.

[0043] (3) Inactivate the enzyme in the enzymatic hydrolysate II by boiling water bath for 25 min and then rapidly cooling to room temperature. Centrifuge at 4000 r / min for 10 min and take the supernatant.

[0044] (4) The peptide content and DPPH free radical scavenging rate of the supernatant of different groups of samples were determined.

[0045] Example 3

[0046] (1) Take 8.0 g of bovine myocardium, clean it, remove connective tissue and fat, homogenize it according to the ratio of bovine myocardium:deionized water = 1:4 (g / mL), sterilize it in boiling water bath for 15 min, cool it rapidly to room temperature, adjust the pH to 11.0, add alkaline protease for enzymatic hydrolysis, and obtain enzymatic hydrolysate I; the enzymatic hydrolysis conditions are temperature 35.0~55.0 ℃, enzyme addition amount 0.70%, and time 2.5 h.

[0047] (2) After cooling the enzymatic hydrolysate I to room temperature, adjust its pH to 7.8, add trypsin for enzymatic hydrolysis to obtain enzymatic hydrolysate II; the enzymatic hydrolysis conditions are temperature 37.0 ℃, time 3.5 h, and enzyme addition amount of 0.09%;

[0048] (3) Inactivate the enzyme in the enzymatic hydrolysate II by boiling water bath for 25 min and then rapidly cooling it to room temperature. Centrifuge at 4000 r / min for 10 min and take the supernatant.

[0049] (4) The peptide content and DPPH free radical scavenging rate of the supernatant of different groups of samples were determined.

[0050] Example 4

[0051] (1) Take 8.0 g of bovine myocardium, clean it, remove fat and connective tissue, homogenize it according to the ratio of bovine myocardium:deionized water = 1:4 (g / mL), sterilize it in boiling water bath for 15 min and cool it rapidly to room temperature, adjust its pH to 11.0, add alkaline protease for enzymatic hydrolysis, and obtain enzymatic hydrolysate I; the enzymatic hydrolysis conditions are temperature 50.0 ℃, enzyme addition amount 0.20~1.50%, and time 2.5 h.

[0052] (2) After cooling the enzymatic hydrolysate I to room temperature, adjust its pH to 7.8, add trypsin for enzymatic hydrolysis to obtain enzymatic hydrolysate II. The enzymatic hydrolysis conditions are: temperature 37.0 ℃, time 3.5 h, and enzyme addition amount of 0.09%.

[0053] (3) Inactivate the enzyme in the enzymatic hydrolysate II by boiling water bath for 25 min and then rapidly cooling it to room temperature. Centrifuge at 4000 r / min for 10 min and take the supernatant.

[0054] (4) The peptide content and DPPH free radical scavenging rate of the supernatant of different groups of samples were determined.

[0055] Example 5

[0056] (1) Take 8.0 g of bovine myocardium, clean it, remove connective tissue and fat, homogenize it according to the ratio of bovine myocardium:deionized water = 1:4 (g / mL), sterilize it in boiling water bath for 15 min, cool it rapidly to room temperature, adjust its pH to 11.0, add alkaline protease for enzymatic hydrolysis, and obtain enzymatic hydrolysate I; the enzymatic hydrolysis conditions are temperature 50.0 ℃, enzyme addition amount 0.70%, and time 2.5 h.

[0057] (2) After cooling the enzymatic hydrolysate I to room temperature, adjust its pH to 7.2-8.5, add trypsin for enzymatic hydrolysis to obtain enzymatic hydrolysate II. The enzymatic hydrolysis conditions are: temperature 37.0 ℃, time 3.5 h, and enzyme addition amount of 0.09%.

[0058] (3) After inactivating the enzyme in the enzymatic hydrolysate II by boiling water bath for 25 min, quickly cool it to room temperature, centrifuge at 4000 r / min for 10 min, and take the supernatant.

[0059] (4) The peptide content and DPPH free radical scavenging rate of the supernatant of different groups of samples were determined.

[0060] Example 6

[0061] Based on the results of single-factor experiments, response surface methodology was conducted to optimize the enzymatic hydrolysis process of bovine myocardial antioxidant peptides. Analysis of variance was performed on the final results using Design-Expert 13.0 software.

[0062] Table 1 Response Surface Factor Level Table

[0063]

[0064] Example 7

[0065] Weigh 15.0 mg of bovine myocardial antioxidant peptide into a 20.0 mL headspace vial, slowly add 10 mL of 1:1 diluted analytical grade hydrochloric acid, tighten the cap, and place in an oven at 110 ℃ for 24 h for hydrolysis. Remove the hydrolyzed sample, cool to room temperature, and filter through a 0.22 μm aqueous membrane into a 25.0 mL volumetric flask, then bring to volume. Pipette 2.0 mL of the diluted sample and deacidify using a nitrogen evaporator at 70.0 ℃ until a small amount of solid or residue remains at the bottom. Add 1.5 mL of sample dilution buffer to the deacidified sample. Mix thoroughly using a shaker. Pipette the sample through a syringe and filter through a 0.22 μm aqueous membrane before analyzing its amino acid composition.

[0066] Example 8

[0067] (1) Take a certain amount of soy protein isolate and 0.2 g of bovine myocardial peptide and dissolve them in 10 mL of water. Stir continuously at room temperature for 3.0 h and then adjust the pH of the solution to 4.0.

[0068] (2) Dissolve a certain amount of sodium alginate in 10 mL of water until the solution is completely transparent; slowly add the soy protein isolate-bovine myocardial peptide solution to the sodium alginate solution, adjust the pH of the mixture to 3.5, and stir continuously at room temperature for 1.0 h; the total mass of soy protein isolate and sodium alginate is 0.3 g, and the mass ratios are 1:0, 4:1, 2:1, 1:1, 1:2, 1:4 and 0:1, respectively;

[0069] (3) After centrifugation, the supernatant was placed at 4 °C for 24 h and then freeze-dried to obtain composite microparticles with soy protein isolate and sodium alginate as carriers.

[0070] (4) Determine the encapsulation efficiency, antioxidant activity and peptide retention rate of the composite microparticles after in vitro gastrointestinal digestion simulation.

[0071] (5) Determination of encapsulation efficiency

[0072] The encapsulation efficiency of bovine myocardial antioxidant peptides encapsulated by composite microparticles was determined by ultraviolet absorption spectrometry. Sample solutions of 10 mg / mL soy protein isolate / sodium alginate at mass ratios of 1:0, 4:1, 2:1, 1:1, 1:2, 1:4, and 0:1 were prepared. 0.75 mL of 5% trichloroacetic acid solution was added to 1.75 mL of each sample solution, and the mixture was allowed to stand at room temperature for 30 min, followed by centrifugation at 6000 r / min for 10 min. Subsequently, 1.0 mL of the supernatant was added to 4.0 mL of biuret reagent, and the mixture was allowed to stand in the dark for 30 min. The absorbance was measured at 540 nm. The content of free peptides in the samples was calculated using a bovine serum albumin standard curve.

[0073] Equation (3)

[0074] In the formula: m is the mass of the free peptide, g; m0 is the mass of the total added peptide, g.

[0075] (6) Antioxidant activity assay

[0076] DPPH free radical scavenging rate determination: Refer to the determination method in Implementation Case 1.

[0077] Ferric ion reducing power determination: 2.0 mL of phosphate buffer (pH 6.6) and 2.5 mL of 1.0% potassium ferricyanide solution were added to 1.0 mL of 10 mg / mL sample solution. After mixing thoroughly, the mixture was reacted in a water bath at 50.0 ℃ for 20 min. After the reaction, the mixture was cooled to room temperature, and 2.5 mL of 10.0% trichloroacetic acid solution was added. After mixing thoroughly, the mixture was centrifuged at 3000 r / min for 10 min. After centrifugation, 2.5 mL of the supernatant was collected and mixed with an equal volume of deionized water. Then, 0.5 mL of 0.1% ferric chloride solution was added, and the mixture was reacted at room temperature for 10 min. The absorbance was measured at 700 nm using a UV spectrophotometer.

[0078] (7) In vitro gastrointestinal digestion simulation

[0079] 10.0 mL of a 10 mg / mL sample solution was mixed thoroughly with an equal volume of simulated gastric juice (SGF). The pH was adjusted to 2.0 with HCl, and pepsin (2.0 mg / mL) and CaCl2 (2.0 mM) were added. The mixture was incubated in a shaker at 37.0 °C and 200 rpm for 2.0 h. A certain amount of the sample solution was taken to determine the content of free peptides in the solution after gastric digestion, and the corresponding volume of simulated gastric juice was added to replace these samples. Next, 20.0 mL of simulated intestinal fluid (SIF) was mixed with the gastric digested sample, and the pH was adjusted to 7.0 with NaOH. Trypsin (2.0 mg / mL), bile salts (10.0 mM), and CaCl2 (0.3 mM) were added. Each sample was incubated at 37.0 °C and 200 rpm for 2.0 h, and the content of free peptides in the solution after gastrointestinal digestion was determined.

[0080] Equation (4)

[0081] In the formula: m is the measured bovine myocardial peptide content (g); M is the total added bovine myocardial peptide content (g).

[0082] Results and Analysis

[0083] (1) According to the results in Example 1, the peptide content (59.477 mg / g) and hydroxyl radical scavenging rate (81.910%) of the bovine myocardial antioxidant peptides obtained by alkaline protease hydrolysis were significantly higher than those obtained by the other three proteases. Simultaneously, the DPPH radical scavenging rate reached 78.356%. Therefore, alkaline protease hydrolysis was the most effective, and the resulting antioxidant peptides exhibited better antioxidant properties. Specific results are shown in Table 2:

[0084] Table 2 Enzymatic hydrolysis results for different types of proteases

[0085]

[0086] (2) The results obtained in Example 2 are attached. Figure 1 As shown, with the increase of alkaline protease hydrolysis time, the content of antioxidant peptides and the DPPH free radical scavenging rate both showed a trend of first increasing and then decreasing; when the hydrolysis time was 2.0 h, the peptide content of the hydrolysis product reached 64.310 mg / mL, and the DPPH free radical scavenging rate was 71.522%, which were the highest compared with other groups.

[0087] (3) The results obtained in Example 3 are shown in the appendix. Figure 2As shown, with the increase of alkaline protease hydrolysis temperature, the content of antioxidant peptides and the DPPH free radical scavenging rate both showed an overall trend of first increasing and then decreasing. When the hydrolysis temperature was 45.0 ℃, the peptide content of the hydrolysis product reached 57.665 mg / mL, and the DPPH free radical scavenging rate was 67.493%, which were the highest compared with other groups, indicating the best hydrolysis effect.

[0088] (4) The results obtained in Example 4 are shown in the appendix. Figure 3 As shown, with the increase of alkaline protease addition, the content of antioxidant peptides and the DPPH free radical scavenging rate showed an overall trend of first increasing and then decreasing; when the alkaline protease addition was 0.70%, the peptide content of the enzymatic hydrolysate was the highest, reaching 72.819 mg / mL; however, when the enzyme addition was 0.50%, the DPPH free radical scavenging rate was the highest, at 66.720%.

[0089] (5) The results obtained in Example 5 are shown in the appendix. Figure 4 As shown, there was no significant difference in the content of antioxidant peptides in the hydrolysate as the pH of trypsin hydrolysis increased; however, the DPPH free radical scavenging rate showed a gradual decreasing trend as the hydrolysis pH increased. At a hydrolysis pH of 7.2 °C, the DPPH free radical scavenging rate was 67.495%, which was the highest among all groups, indicating the best hydrolysis effect.

[0090] (6) In Example 6, the enzymatic hydrolysis preparation process of bovine myocardial antioxidant peptides was optimized using response surface methodology to ensure that the antioxidant capacity of the final enzymatic hydrolysis product was optimal. Specific results are shown in Tables 3 and 4 and Appendix. Figure 5 As shown

[0091] Table 3 Response Surface Experimental Design and Results

[0092]

[0093] Table 4 Results of Analysis of Variance

[0094]

[0095] * indicates a significant difference (0.01 < P < 0.05); ** indicates a very significant difference (0.001 < P < 0.01); *** indicates an extremely significant difference (P < 0.001).

[0096] The experimental results were analyzed using Design-Expert 13.0 software to obtain the standard regression equation for the DPPH free radical scavenging rate:

[0097] Y=85.35-0.6144A-0.7457B-0.1822C-3.56D-1.04AB+2.56AC-4.25AD-0.6687BC-1.91BD-1.07CD-4.00A 2 -2.97B 2 -2.62C 2 -6.87D 2

[0098] As shown in Table 4, the quadratic equation model exhibits significant differences (P < 0.0001), and the degree of lack of fit of the regression equation, P = 0.8396 > 0.05, indicates that the lack of fit test result is not significant, suggesting that the quadratic regression model has a good fit and small experimental error. Correlation coefficient R0 2 =0.982, demonstrating a high correlation between the experimental and predicted values. Meanwhile, the correction coefficient R0... 2 Adj =0.962, proving that the model can explain 96.20% of the response value variation. The model has high reliability and can be used as a basis for further analysis.

[0099] Response surface methodology analysis revealed the optimal preparation conditions for bovine myocardial antioxidant peptides as follows: alkaline protease hydrolysis time of 2.06 h, hydrolysis temperature of 44.70 ℃, enzyme addition of 0.47%, and trypsin hydrolysis pH of 7.41. Under these conditions, three replicate experiments were conducted for validation. The average DPPH free radical scavenging rate of the prepared bovine myocardial antioxidant peptides was measured to be 85.431%, which is close to the predicted value of 85.85%, demonstrating the reliability of the model.

[0100] (7) The amino acid composition of the bovine myocardial antioxidant peptides obtained in Example 7 is shown in Table 5. The antioxidant peptides were determined to be mainly composed of 18 amino acids, including 7 essential amino acids. Among these 18 amino acids, glutamic acid had the highest proportion, accounting for 18.450% of the total amino acids; followed by lysine and aspartic acid, accounting for 10.680% and 10.574% of the total amino acids, respectively.

[0101] Table 5 Amino acid composition of bovine myocardial antioxidant peptides

[0102]

[0103] (8) The results obtained in Example 8 are attached. Figure 6-9As shown in the figure, compared with a soy protein isolate / sodium alginate mass ratio of 1:0, the encapsulation efficiency of composite microparticles with different soy protein isolate / sodium alginate mass ratios increased to varying degrees. The results indicate that the presence of sodium alginate enhances the ability of soy protein isolate particles to encapsulate and retain bovine myocardial antioxidant peptides during antisolvent precipitation. However, compared with bovine myocardial antioxidant peptides, the antioxidant activity of the same amount of composite microparticles was reduced. Furthermore, during gastrointestinal digestion, the retention rate of bovine myocardial antioxidant peptides in the soy protein isolate / sodium alginate mass ratios of 2:1, 1:1, 1:2, and 1:4 was significantly higher than that in the control group, and the 1:0 and 0:1 groups. This result indicates that encapsulating bovine myocardial antioxidant peptides with soy protein isolate and sodium alginate can effectively protect them from digestion and degradation in the gastrointestinal tract and improve their utilization rate.

[0104] (9) Overall, the DPPH free radical scavenging rate of the bovine myocardial antioxidant peptide obtained in this invention is as high as 85.431%, and it can be used as a natural antioxidant peptide in the fields of food, medicine and skin care products. Moreover, the encapsulation of bovine myocardial peptide with soy protein isolate / sodium alginate makes the structure of the composite microparticles more stable, significantly reduces the degree of digestion and degradation of bovine myocardial peptide in the gastrointestinal tract, and improves the utilization rate of bovine myocardial peptide in the body.

Claims

1. A method for preparing bovine myocardial antioxidant peptides and their composite microparticles, characterized in that, Includes the following steps: Preparation of bovine myocardial antioxidant peptides: Pretreatment: Fresh bovine heart muscle is selected as raw material. Fat and connective tissue are removed, and blood is rinsed off. Then, a certain proportion of deionized water is added for homogenization to obtain raw material solution. One-time enzymatic hydrolysis: After boiling the raw material solution for 15 minutes to sterilize it, cool it to room temperature, adjust the pH value to a suitable range, add protease and carry out enzymatic hydrolysis under suitable temperature and time conditions to obtain enzymatic hydrolysate I; Secondary enzymatic hydrolysis: Cool the enzymatic hydrolysate I to room temperature, adjust the pH value to a suitable range, add an appropriate amount of trypsin to continue the enzymatic hydrolysis reaction, and obtain enzymatic hydrolysate II; Enzyme inactivation: The enzyme hydrolysate II was inactivated and centrifuged. The supernatant was then freeze-dried to obtain bovine myocardial antioxidant peptide powder. Preparation of composite microparticles: Take a certain amount of soy protein isolate and bovine myocardial peptide and dissolve them in 10 mL of deionized water. After stirring continuously at room temperature for a certain period of time, adjust the pH of the solution to a suitable value. Dissolve a certain amount of sodium alginate in 10 mL of deionized water and stir until the solution is completely transparent. Soy protein isolate-bovine cardiac peptide solution was slowly added to sodium alginate solution. The pH of the mixed solution was adjusted to a suitable value. The mixture was stirred continuously at room temperature for a certain period of time. After centrifugation, the supernatant was collected and placed at 4.0 ℃ for 24 h before freeze-drying to obtain composite microparticle powder.

2. The method for preparing bovine myocardial antioxidant peptides and their composite microparticles according to claim 1, characterized in that, The pretreatment described in 1.1.1 includes homogenizing bovine heart with deionized water at a solid-liquid ratio of 1:4 g / mL to obtain bovine myocardial raw material solution.

3. The method for preparing bovine myocardial antioxidant peptides and their composite microparticles according to claim 1, characterized in that, In the first enzymatic hydrolysis in section 1.1.2, alkaline protease, papain, flavor protease, and pepsin were used for hydrolysis, with alkaline protease showing the best hydrolysis effect. When using alkaline protease for hydrolysis, the pH of the sterilized raw material solution was adjusted to 8.0~12.0 before hydrolysis, the amount of alkaline protease added was 0.20~1.50% of the weight of bovine myocardium, the hydrolysis time was 1.5~3.5 h, and the hydrolysis temperature was 35.0~55.0 ℃.

4. The method for preparing bovine myocardial antioxidant peptides and their composite microparticles according to claim 1, characterized in that, In step 1.1.3, before the second enzymatic hydrolysis, the pH of the hydrolysate I is adjusted to 7.2-8.5, the amount of trypsin added is 0.05-0.20% of the bovine myocardium mass, and the hydrolysis time is 2.5-4.5 h.

5. Prepare bovine myocardial antioxidant peptides according to the preparation method described in claims 1-4.

6. The method for preparing bovine myocardial antioxidant peptide and its composite microparticles according to claim 5, characterized in that, The peptide content of the enzymatic hydrolysate ranges from 45.88 to 72.82 mg / g, the DPPH radical scavenging capacity ranges from 27.83 to 82.73%, and the hydroxyl radical scavenging rate ranges from 40.25 to 81.91%. After optimization by response surface methodology, the DPPH radical scavenging capacity of the enzymatic hydrolysate can reach 85.43%.

7. The method for preparing bovine myocardial antioxidant peptides and their composite microparticles according to claim 1, characterized in that, In step 1.2.1, the amount of bovine myocardial peptide added was 0.2 g, the stirring time was 3.0 h, and the pH value of the solution was 4.

0.

8. The method for preparing bovine myocardial antioxidant peptides and their composite microparticles according to claim 1, characterized in that, The solution described in 1.2.3 has a pH of 3.5 and a stirring time of 1.0 h.

9. The method for preparing bovine myocardial antioxidant peptide and its composite microparticles according to claim 1, characterized in that, In 1.2, the total weight of soy protein isolate / sodium alginate was 0.3 g, with mass ratios of 1:0, 4:1, 2:1, 1:1, 1:2, 1:4 and 0:1, respectively.

10. The preparation method according to claims 7-9 is used to prepare composite microparticles loaded with bovine myocardial antioxidant peptides.

11. The method for preparing bovine myocardial antioxidant peptides and their composite microparticles according to claim 10, characterized in that, The encapsulation efficiency of the composite microparticles was 57.01~72.79%, the DPPH free radical scavenging capacity was 45.25~63.24%, the absorbance value of the iron ion reducing power was 0.12~0.20, and the peptide retention rate after in vitro gastrointestinal digestion was 20.76~27.52%.

Images