Preparation method and application of a buffalo colostrum milk source active peptide

Abstract

The present invention relates to the technical field of whey protein hydrolysis, and particularly to a preparation method and application of buffalo colostrum-derived bioactive peptides. The present invention optimizes the enzymatic hydrolysis process of buffalo colostrum whey protein through single-factor experiments to obtain an enzymatic hydrolysis method of buffalo colostrum whey protein suitable for large-scale production. In addition, the enzymatic hydrolysis process of buffalo colostrum whey protein is optimized by the response surface method. Three factors, namely the pH value, enzymatic hydrolysis time, and enzyme addition amount during enzymatic hydrolysis with neutral protease, are selected as corresponding variables, and the ABTS radical scavenging rate is used as the response value. Multiple fittings are performed on each factor to obtain the optimal enzymatic hydrolysis process conditions for whey protein. The buffalo colostrum-derived bioactive peptides obtained by this enzymatic hydrolysis method have the technical advantages of high ABTS radical scavenging rate and good immune function. At the same time, the stability of the enzymatic hydrolysis product is further studied through gastrointestinal digestion experiments, providing a more comprehensive theoretical reference for the further research of whey protein.

Description

Technical Field

[0001] The present invention relates to the technical field of whey protein hydrolysis, and particularly relates to a preparation method and application of buffalo colostrum-derived bioactive peptides.

Background Art

[0002] Milk-derived bioactive peptides are specific protein fragments composed of 2 - 50 amino acid sequences derived from milk proteins and have different physiological functions. Due to their characteristics such as high specificity, low toxicity, high structural diversity, and low molecular weight, they are considered ideal substances for use in health products or functional foods.

[0003] Milk-derived bioactive peptides have various physiological functions such as antioxidant, antibacterial, regulating the body's immunity, lowering blood pressure, anti-inflammatory, anti-cancer, and regulating metabolic syndrome. Among them, the antioxidant property of milk-derived peptides is a relatively widely studied aspect. The potential side effects of synthetic antioxidants are controversial, so people prefer to choose natural low-toxic antioxidants.

[0004] Milk proteins contain many peptide sequences with different biological activities, but these bioactive peptides can only play their roles after being released by some means. There are mainly three methods for obtaining bioactive peptides from milk proteins: fermentation, obtaining them by fermenting milk proteins with microorganisms; enzymatic hydrolysis, obtaining them by hydrolyzing milk proteins with endogenous or exogenous proteases; and biosynthesis, synthesizing according to the sequences of bioactive peptides.

[0005] However, there is currently relatively little research on the release of immune-active peptides from buffalo milk proteins. Buffalo milk is the second largest milk in the world, with its production accounting for about 13% of the global milk production, and China is a major producer of buffalo milk. Compared with the milk of other mammals, the protein content in Chinese buffalo milk is as high as 4.3%, and that in buffalo colostrum even reaches 10%. Whey protein is one of the main products in the dairy industry, accounting for about 80% of buffalo colostrum, but its proportion will decrease with the prolongation of lactation time. This provides a raw material guarantee for the preparation of high-quality milk-derived bioactive peptides. Therefore, it has important economic significance to study the development and preparation of buffalo milk protein immune-active peptides using buffalo colostrum proteins.

Summary of the Invention

[0006] In view of the above, it is necessary to provide a method for efficiently preparing buffalo colostrum-derived bioactive peptides, which can better hydrolyze the whey protein in buffalo colostrum and provide an effective research basis for the application of buffalo colostrum-derived bioactive peptides.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for preparing buffalo colostrum-derived bioactive peptides, and the enzymatic hydrolysis method is as follows:

[0009] (1) Adjust the pH value of buffalo colostrum to (4.6 ± 0.2), and centrifuge at (4 ± 1) °C to remove the upper layer of fat and precipitate, obtaining a buffalo colostrum whey protein solution;

[0010] (2) After adjusting the pH value of the enzymolysis solution of buffalo colostrum whey protein solution obtained in step (1), preheat for 8 - 10 minutes and then add neutral protease for constant-temperature enzymolysis to obtain an enzymolysis solution;

[0011] (3) Centrifuge the enzymolysis solution in step (2), take the supernatant, and adjust the pH to neutral to obtain the buffalo colostrum milk source active peptide;

[0012] In the said step (2), the pH value is 6.5 - 7.5, the enzymolysis time of neutral protease is 2 - 4 h, and the enzyme addition amount during enzymolysis is 2000 - 4000 U / g.

[0013] Further, in the said step (2), the pH value is 7.5, the enzymolysis time of neutral protease is 2.0 h, and the enzyme addition amount during enzymolysis is 6299 U / g.

[0014] Further, in the said step (1), the centrifugation condition is centrifuging at 5000 ± 500 r / min for 20 min.

[0015] Further, in the said step (3), the centrifugation condition is centrifuging at 8000 ± 500 r / min for 15 ± 2 min

[0016] Further, in the said steps (1) to (3), the reagent used for adjusting the pH value is 1 M sodium hydroxide solution or 1 M hydrochloric acid solution.

[0017] The present invention also includes the application of the buffalo colostrum milk source active peptide prepared by the said preparation method in the preparation of foods or drugs related to improving cell proliferation rate, cell phagocytosis rate or cell immunity.

[0018] The present invention also includes a method for preparing the said food or drug, and the said method includes the following steps:

[0019] (1) Adjust the pH value of buffalo colostrum to (4.6 ± 0.2), and centrifuge at (4 ± 1) °C to remove the upper layer of fat and precipitate, obtaining a buffalo colostrum whey protein solution;

[0020] (2) After adjusting the pH value of the enzymolysis solution of buffalo colostrum whey protein solution obtained in step (1), preheat for 8 - 10 minutes and then add neutral protease for constant-temperature enzymolysis to obtain an enzymolysis solution;

[0021] (3) Centrifuge the enzymolysis solution in step (2), take the supernatant, and adjust the pH to neutral to obtain the buffalo colostrum milk source active peptide;

[0022] (4) Freeze-dry the buffalo colostrum milk source active peptide obtained in step (3) and then perform enteric coating to obtain the said food or medicine;

[0023] In the said step (2), the pH value is 6.5 - 7.5, the enzymolysis time of neutral protease is 2 - 4 h, and the enzyme addition amount during enzymolysis is 5000 - 7000 U / g.

[0024] Furthermore, the enteric coating is prepared by mixing ethylcellulose aqueous dispersion, talcum powder, triethyl citrate and polyethylene glycol in a mass ratio of 10:2:1:0.1.

[0025] The present invention has the following beneficial effects:

[0026] 1. The present invention optimizes the enzymolysis process of buffalo colostrum whey protein through single-factor experiments to obtain a method for enzymolyzing buffalo colostrum whey protein suitable for large-scale production; in addition, the enzymolysis process of buffalo milk whey protein is also optimized by the response surface method. Three factors, namely the pH value, enzymolysis time and enzyme addition amount during enzymolysis of neutral protease, are selected as corresponding variables, and the ABTS free radical scavenging rate is used as the response value. Multiple fittings are carried out on each factor to obtain the optimal enzymolysis process conditions of whey protein; the buffalo colostrum milk source active peptide obtained by enzymolysis with this method has the technical advantages of high ABTS free radical scavenging rate and good immune function. At the same time, the stability of the enzymolysis product is further studied through gastrointestinal digestion experiments, providing a more comprehensive theoretical reference for the further research of whey protein.

Description of the Drawings

[0027] Figure 1 It is a response surface diagram of the influence of the ABTS free radical scavenging rate of milk source active peptide under the interaction of pH and time;

[0028] Figure 2 It is a response surface diagram of the influence of the ABTS free radical scavenging rate of milk source active peptide under the interaction of pH and enzyme addition amount;

[0029] Figure 3 It is a response surface diagram of the influence of the ABTS free radical scavenging rate of milk source active peptide under the interaction of time and enzyme addition amount;

[0030] Figure 4 It is a contour diagram of the influence of the ABTS free radical scavenging rate of milk source active peptide under the interaction of pH and time;

[0031] Figure 5 It is a contour diagram of the influence of the ABTS free radical scavenging rate of milk source active peptide under the interaction of pH and enzyme addition amount;

[0032] Figure 6Contour plot of the effect of interaction between time and enzyme addition amount on the ABTS radical scavenging rate of milk-derived bioactive peptides;

[0033] Figure 7 Result graph of the effect of the optimal milk-derived bioactive peptides on the cell proliferation rate of macrophages RAW264.7;

[0034] Figure 8 Result graph of the effect of the optimal milk-derived bioactive peptides on the phagocytosis rate of macrophages RAW264.7;

[0035] Figure 9 Result graph of the effect of milk-derived bioactive peptides after simulated gastrointestinal digestion on the cell proliferation rate of macrophages RAW264.7;

[0036] Figure 10 Result graph of the effect of milk-derived bioactive peptides after simulated gastrointestinal digestion on the phagocytosis rate of macrophages RAW264.7;

[0037] Figure 11 Result graph of the effect of milk-derived bioactive peptides after simulated gastrointestinal digestion on the NO release amount of macrophages RAW264.7;

[0038] Figure 12 Result graph of the effect of milk-derived bioactive peptides after simulated gastrointestinal digestion on the ABTS radical scavenging rate;

[0039] Figure 13 Result graph of the effect of milk-derived bioactive peptides after simulated gastrointestinal digestion on the reducing power;

[0040] Figure 14 Result graph of the effect of enzymatic hydrolysates with different enzyme addition amounts on the cell proliferation rate of macrophages RAW264.7;

[0041] Figure 15 Result graph of the effect of enzymatic hydrolysates with different enzyme addition amounts on the phagocytosis rate of macrophages RAW264.7;

[0042] Figure 16 Result graph of the effect of enzymatic hydrolysates with different enzyme addition amounts after simulated gastrointestinal digestion on the cell proliferation rate of macrophages RAW264.7;

[0043] Figure 17 Result graph of the effect of enzymatic hydrolysates with different enzyme addition amounts after simulated gastrointestinal digestion on the phagocytosis rate of macrophages RAW264.7;

[0044] Figure 18 Result graph of the effect of enzymatic hydrolysates with different enzyme addition amounts after simulated gastrointestinal digestion on the NO release amount of macrophages RAW264.7;

[0045] Figure 19Graph showing the effect of simulated gastrointestinal digestion on the ABTS radical scavenging rate of enzymolysis products with different enzyme addition amounts;

[0046] Figure 20 Graph showing the effect of simulated gastrointestinal digestion on the reducing power of enzymolysis products with different enzyme addition amounts.

Specific Embodiments

[0047] All features disclosed in this specification, or all steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any manner.

[0048] Any feature disclosed in this specification (including any additional claims, abstract) is, unless otherwise stated, only an example of a series of equivalent or similar features.

[0049] Process conditions:

[0050] A method for preparing a whey protein hydrolysate (milk source active peptide) from buffalo colostrum, characterized by sequentially performing the following steps:

[0051] 1. Preparation of buffalo colostrum whey protein:

[0052] Adjust the pH value of buffalo colostrum to (4.6 ± 0.2), and centrifuge at (4 ± 1) °C to remove the upper layer of fat and precipitate, obtaining a buffalo colostrum whey protein solution; in this step, the centrifugation conditions are: centrifuge at (5000 ± 500) r / min for 20 min.

[0053] 2. Protease enzymolysis:

[0054] (1) Adjust the pH value of the buffalo colostrum whey protein solution, preheat at (40 ± 2) °C for 8 - 10 minutes, then add neutral protease for constant temperature enzymolysis (i.e., the enzymolysis temperature is the same as the above preheating temperature), and the enzymolysis time is 0.5 - 4 h to obtain an enzymolysis solution;

[0055] Among them, the addition amount of neutral protease is: 2000 - 5500 U / g of buffalo colostrum whey protein (as the substrate); after the enzymolysis time is up, terminate the enzymolysis by boiling water bath for 10 min to inactivate the enzyme;

[0056] (2) Centrifuge the enzymolysis solution, take the supernatant, and adjust the pH to neutral to obtain the milk source active peptide. In this step, the centrifugation conditions are: centrifuge at (8000 ± 500) r / min for (15 ± 2) min.

[0057] In this process, 1M sodium hydroxide solution / 1M hydrochloric acid solution is used to adjust the pH value;

[0058] The present invention uses the antioxidant capacity of the enzymolysis solution as the main evaluation criterion for the product performance.

[0059] In the present invention, the neutral protease can be purchased from Shanghai Macklin Biochemical Co., Ltd., and its enzyme activity is 50 U / mg.

[0060] The enzymatic hydrolysis technical route of the present invention is as follows:

[0061] Buffalo colostrum → adjust pH to 4.6 → centrifuge → defat and take supernatant → adjust pH → heat → inactivate enzyme in boiling water bath → adjust pH to neutral → centrifuge → take supernatant.

[0062] Example 1:

[0063] This example mainly studies the optimal hydrolysis conditions for the enzymatic hydrolysis process of buffalo colostrum whey by the response surface method to prepare buffalo colostrum-derived bioactive peptides with enhanced immune effects.

[0064] I. The specific preparation method for the enzymatic hydrolysis of buffalo colostrum whey includes the following steps:

[0065] 1. Adjust the pH value of buffalo colostrum to 4.6, centrifuge at 5000 r / min for 20 min at 4 °C to remove the upper layer of fat and precipitate, and obtain the buffalo colostrum whey protein solution.

[0066] 2. Adjust the pH value of the buffalo colostrum whey protein solution to 7.0, preheat at 40 °C for 10 minutes, then add 5000 U / g of neutral protease for constant-temperature enzymatic hydrolysis (i.e., the enzymatic hydrolysis temperature is the same as the above preheating temperature), and the enzymatic hydrolysis time is 60 min to obtain the enzymatic hydrolysate. After the enzymatic hydrolysis time is up, inactivate the enzyme in a boiling water bath for 10 min to end the enzymatic hydrolysis;

[0067] 3. Centrifuge the enzymatic hydrolysate at 8000 r / min for 15 min, take the supernatant, and adjust the pH to neutral to obtain buffalo colostrum-derived bioactive peptides.

[0068] In this example, the pH value is adjusted using 1 M sodium hydroxide solution or 1 M hydrochloric acid solution;

[0069] In this example, the neutral protease can be purchased from Shanghai Macklin Biochemical Co., Ltd., and its enzyme activity is 50 U / mg. In this example, the antioxidant capacity of buffalo colostrum-derived bioactive peptides is used as the main evaluation criterion for the product performance. Among them, the measurement method for antioxidant properties is as follows:

[0070] Dilute the ABTS stock solution 400 times, adjust it to make the OD value at 405 nm be 0.7. Dilute the enzymatic hydrolysis substrate 10 times. Add 50 μl of the diluted enzymatic hydrolysis substrate solution and 200 μl of the ABTS working solution to a 96-well plate. Use water instead of the diluted enzymatic hydrolysis substrate solution as a blank control. After mixing, react in the dark at 37 °C for 30 min, and measure its OD value at 405 nm. Calculate the ABTS radical scavenging rate according to the following formula:

[0071] ABTS radical scavenging rate (%) = (A 空白 - A 样品 ) / A 空白 × 100%;

[0072] In the formula for calculating the ABTS radical scavenging rate: A blank is the OD value of the blank control at 405 nm; A sample is the OD value of the buffalo colostrum-derived bioactive peptide at 405 nm.

[0073] In this example, the ABTS radical scavenging rate of the buffalo colostrum-derived bioactive peptide is 86.16%.

[0074] Example 2:

[0075] The specific operation process is the same as that of Example 1, except that different enzymes are used: pepsin, trypsin, alkaline protease, papain, and neutral protease for enzymatic hydrolysis. The ABTS radical scavenging rates of the buffalo colostrum-derived bioactive peptides finally measured are shown in Table 1:

[0076] Table 1 Antioxidant properties of buffalo colostrum-derived bioactive peptides obtained by hydrolysis with different enzymes

[0077] Enzyme type ABTS radical scavenging rate / % Pepsin 63.66 Trypsin 82.94 Alkaline protease 84.00 Papain 70.76 Neutral protease 86.16

[0078] As can be seen from Table 1, among the above various enzymes, the antioxidant property of the bioactive peptide derived from buffalo colostrum whey protein obtained by enzymatic hydrolysis with neutral protease is the highest, reaching 86.16%.

[0079] Example 3:

[0080] The specific operation process is the same as that of Example 1, except for the basic conditions of enzymatic hydrolysis: the pH of enzymatic hydrolysis is 6, 6.5, 7, 7.5, 8, the enzyme addition amount is 4000 U / g, and the enzymatic hydrolysis time is 1 h. The ABTS radical scavenging rates of the buffalo colostrum-derived bioactive peptides finally measured are shown in Table 2:

[0081] Table 2 Effect of enzymatic hydrolysis pH on the antioxidant property of buffalo colostrum-derived bioactive peptides

[0082]

[0083] As can be seen from Table 2, at the same time and enzyme addition amount, the ABTS radical scavenging rate of the buffalo colostrum-derived bioactive peptide first increases and then decreases with the increase of pH.

[0084] Example 4:

[0085] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the pH of enzymatic hydrolysis is 7, the enzyme addition amount is 4000 U / g, and the enzymatic hydrolysis times are 1, 2, 3, 4, and 5 h. The ABTS radical scavenging rates of the bioactive peptides from buffalo colostrum finally measured are shown in Table 3:

[0086] Table 3 Effects of enzymatic hydrolysis time on the antioxidant properties of bioactive peptides from buffalo colostrum

[0087]

[0088] As can be seen from Table 3, at the same pH and enzyme addition amount, the ABTS radical scavenging rate of the bioactive peptides from buffalo colostrum first increases and then remains unchanged with the increase of time.

[0089] Example 5:

[0090] The specific operation process is the same as that in Example 1, except for the basic conditions of enzymatic hydrolysis: the pH of enzymatic hydrolysis is 7, the enzyme addition amounts are 2000, 3000, 4000, 5000, and 6000 U / g, and the enzymatic hydrolysis time is 1 h. The ABTS radical scavenging rates of the bioactive peptides from buffalo colostrum finally measured are shown in Table 4:

[0091] Table 4 Effects of enzyme addition amount on the antioxidant properties of bioactive peptides from buffalo colostrum

[0092]

[0093] As can be seen from Table 4, at the same pH and time, the ABTS radical scavenging rate of the bioactive peptides from buffalo colostrum first increases and then decreases with the increase of enzyme addition amount.

[0094] Example 6:

[0095] Response surface optimization analysis:

[0096] According to the central composite experimental design principle of Box-Behnken response surface design, combined with the above experimental results, three influencing factors of enzymatic hydrolysis pH, enzymatic hydrolysis time, and enzyme addition amount are selected, each taking three levels, and the response surface analysis method of three factors and three levels is used to optimize the hydrolysis conditions.

[0097] Table 5 Coding table of three factors and three levels

[0098]

[0099] Table 6 Response surface experimental design and results

[0100]

[0101] The experimental results in Table 6 were subjected to quadratic multiple regression fitting using Design Expert software, and an analysis of variance was performed. The results are shown in Table 7. The significance of the influence of each variable on the response value in the regression equation is determined by the F-test. The smaller the probability P value, the higher the significance of the response variable. P less than 0.05 indicates that the variable term is significant, and greater than 0.1 represents that the variable term is not significant.

[0102] Table 7 Response surface analysis of variance

[0103]

[0104]

[0105] As can be seen from Table 7, the F value of the quadratic term model selected in this experiment is 61.69, which is significant (P < 0.01). Among all variables, the P values corresponding to X1, X3, X1X2, X1X3, X12, X22, and X32 are all less than 0.05, indicating that they have a significant impact on the experimental results. The F value of the lack-of-fit term is 2.45, which is not significant, indicating that the model is reliable. The multiple correlation coefficient R2 is 0.9875, which indicates that 98.75% of the change in the degree of hydrolysis comes from the selected variables. Therefore, the regression equation can well describe the true relationship between each variable and the response value, and the optimal hydrolysis conditions can be determined using this regression equation. In addition, the model established when the signal-to-noise ratio is greater than 4 is credible. The signal-to-noise ratio in this experiment is 29.9079, which is much greater than 4, further indicating that the experimental model is reliable and can well reflect the relationship between the ABTS radical scavenging rate and pH, enzyme dosage, and time. Within the selected factor level range, the influence of each variable on the experimental results is in the order of: pH > enzyme dosage > time. By fixing the value of one variable and examining the influence of the other two variables on the ABTS radical scavenging rate, the response surface diagram is drawn. The influence results of pH, enzyme dosage, and time on the ABTS radical scavenging rate are shown in Figures 1 - 6 . As the variable changes, the ABTS radical scavenging rate first shows an upward trend and then starts to decline after reaching the highest point. According to the literature report, the contour line at the bottom of the response surface presents an ellipse, indicating that the interaction of the variables is significant. The experimental results show that the interaction between pH and enzyme dosage is the strongest. The response surface diagram shows good accuracy in predicting the significant interaction between the degree of hydrolysis and the two combined variables, and the results are consistent with the regression model.

[0106] Using the Design-Expert 11 statistical software to solve the equation, it is obtained that under the conditions of neutral protease hydrolysis, the optimal hydrolysis process of whey protein is as follows: pH value 7.5, hydrolysis time of neutral protease 2.0 h, enzyme addition amount of 6299 U / g during hydrolysis. Under this condition, the predicted scavenging rate of ABTS free radicals is 90.55%, and the actual scavenging rate of ABTS free radicals is 90.37%, which is very close to the theoretical predicted value. Therefore, the neutral protease hydrolysis conditions optimized by the central composite experimental design of Box-Behnken are accurate and reliable, and have practical value.

[0107] Example 7:

[0108] Enhanced cell immunity experiment:

[0109] 1. Cell resuscitation: Prepare a 15 ml centrifuge tube and a T-25 culture flask in the sterile area and add 5 ml of complete medium to each; place the cryopreservation tube in a 37 °C water bath, hold the cryopreservation tube and shake it constantly until the contents are completely melted. Then immediately remove the cryopreservation tube from the water bath, dry it and spray 75% ethanol, and transfer it to the sterile area; carefully remove the lid without touching the internal threads, gently aspirate the cell suspension with a pipette, add it to the prepared 15 ml centrifuge tube, and centrifuge at 1000 rpm for 5 min; after discarding the supernatant, gently flick the bottom of the centrifuge tube to disperse the cell pellet, add an appropriate amount of complete medium to resuspend the cells, and then transfer them to the prepared T25 culture flask; gently shake the culture flask to evenly distribute the cells; place the culture flask in a CO2 incubator for culturing. This cell passage does not require digestion with trypsin. When passing the cells, aspirate part of the culture medium, leave a little culture medium, scrape the cell surface with a sterile cell scraper to scrape off the cells, and inoculate them into a new culture flask filled with fresh culture medium after pipetting.

[0110] 2. Cell passage: After aspirating the supernatant (the cells in the supernatant should also be collected, and these floating cells are viable), wash once with PBS, add 7 - 8 ml of complete medium, scrape the cell surface with a sterile cell scraper to scrape off the cells, and gently pipette and inoculate them into a new culture flask filled with fresh culture medium.

[0111] 3. Cell culture (96-well plate): Scrape the cells with a cell scraper, pipette and mix well, take 10 ul of the cell suspension and add it to a hemocytometer to calculate the cell concentration. The number of cells = (W1 + W2 + W3 + W4) / 4 × 10 4 × dilution ratio, calculate the dilution multiple V1 × M1 = V2 × M2, and add to the 96-well plate at 10 4 cells / 100 ul / well and culture for 24 h.

[0112] 4. Sample Intervention: Remove the culture medium, add the sample and culture medium (sample: culture medium = 100 ul: 100 ul, total 200 ul), set up negative control (100 ul water), positive control (LPS: 100 ng / ml), and intervene for 24 h;

[0113] 5. Detection of Cell Proliferation Rate: Intervene and culture the cells according to steps 3 and 4; Take the culture medium into a 2 ml centrifuge tube for testing; Add 100 ul of culture medium and 10 μl of CCK-8 Solution to each well; Add 3 blank groups (100 ul of culture medium), and also add 10 μl of CCK-8 Solution; Incubate in the cell culture incubator for 4 h; Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader;

[0114] Conversion formula: Percentage of cell survival = [(A - C) / (B - C)] × 100%

[0115] A: Absorbance value of the experimental group (the absorbance value containing culture medium, cells, test drug, and CCK-8 Solution)

[0116] B: Absorbance value of the control group (the absorbance value containing culture medium, cells, and CCK-8 Solution)

[0117] C: Absorbance value of the blank group (the absorbance value containing culture medium and CCK-8 Solution)

[0118] 6. Determination of Cell Phagocytic Activity by Neutral Red Staining Method: Intervene and culture the cells according to steps 3 and 4; Take the culture medium into a 2 ml centrifuge tube for testing; Add 100 μL of neutral red solution with a concentration of 0.1%, and continue to culture for 4 h. Discard the neutral red solution, wash 3 times with PBS, then add 200 μL of cell lysate to each well. The blank group only adds 200 μL of cell lysate, and let it stand overnight at 4 °C, and measure the OD value at a wavelength of 540 nm.

[0119] Phagocytosis rate / % = (A 样品组 - A 空白组 ) / (A 对照组 - A 空白组 ) × 100%;

[0120] The results of cell proliferation rate and cell phagocytosis rate indexes are shown in detail in Figure 3 、 Figure 4 ;

[0121] The proliferation activity of macrophages RAW264.7 after being treated with buffalo colostrum-derived bioactive peptides obtained by enzymatic hydrolysis under the optimal enzymatic hydrolysis conditions obtained by neutral protease in response surface optimization for 24 h is as Figure 3As shown in the figure, BWD in the figure is the bioactive peptide from buffalo colostrum. The results showed that after the intervention of BWD, the cell proliferation rate increased significantly compared with the control group, reaching 118.82%.

[0122] After the bioactive peptide from buffalo colostrum obtained by enzymatic hydrolysis with neutral protease under the optimal enzymatic hydrolysis conditions obtained by response surface optimization was used to treat macrophages RAW264.7, the phagocytic activity after 24 hours was as Figure 4 shown in the figure. BWD in the figure is the bioactive peptide from buffalo colostrum, and BW is the whey protein from buffalo colostrum. The results showed that after the intervention of BWD and BW, the phagocytosis rate of cells increased compared with the control group, and the phagocytic activity was significantly higher than that of the control group and the BW group after the intervention of BWD.

[0123] In in vitro experiments, the proliferation and phagocytosis of macrophages RAW264.7 were used to evaluate the immunomodulatory effect of the enzymatic hydrolysate of buffalo colostrum (bioactive peptide from buffalo colostrum). The results showed that the cell proliferation rate and phagocytosis rate after the intervention of BWD were significantly higher than those of the control group, and the phagocytic activity was significantly higher than that of the BW group after the intervention of BWD, indicating that the bioactive peptide from buffalo colostrum has an immune-enhancing effect.

[0124] Example 8:

[0125] Simulated artificial gastrointestinal experiment:

[0126] 1. Preparation of artificial gastrointestinal fluid

[0127] Table 8 Components of artificial gastrointestinal fluid

[0128]

[0129]

[0130] Prepare the stock solutions of artificial simulated gastric fluid (SGF) and artificial simulated intestinal fluid (SGF) according to the table for standby.

[0131] 2. Simulated gastric digestive system: Take 10 mL of the enzymolysis substrate solution, add 8 mL of the SGF stock solution, adjust the pH to 3.0 with 1.0 mol / L HCl solution, then add pepsin and 5 μL of 0.3 mol / L CaCl2, and finally add ultrapure water to make the system volume up to 20 mL. Incubate in a constant temperature shaker at 37 °C for 120 min. (2) Simulated intestinal digestive system: Take 20 mL of the gastric chyme after gastric digestion, add 16 mL of the SIF stock solution, adjust the pH to 7.0 with 1.0 mol / L NaOH solution, add trypsin, 10 mg / ml porcine bile salt, 20 μL of 0.3 mol / L CaCl2, and finally make up the system volume to 40 mL with ultrapure water. After incubating in a constant temperature shaker at 37 °C for 120 min, inactivate the enzyme in a boiling water bath for 10 min to terminate the reaction. Store at -20 °C in a refrigerator for later use.

[0132] 3. Immunological activity of simulated gastrointestinal digest

[0133] The results of the cell proliferation rate of the simulated gastrointestinal digestion products are shown in detail in Figure 9 ;

[0134] The proliferation activity of the water buffalo colostrum-derived bioactive peptides obtained by enzymolysis under the optimal enzymolysis conditions obtained by neutral protease in response surface optimization and their simulated gastrointestinal digestion products on macrophages RAW264.7 after 24 h is as shown in Figure 9 shown. In the figure, BWD is the water buffalo colostrum-derived bioactive peptide. The results showed that after intervention and culture with BWD, the cell proliferation rate was significantly increased compared with the control group. After BWD was simulated gastric digested, the cell proliferation rate decreased but still increased compared with the control. After the simulated gastric digestion products were further intestinal digested, the cell proliferation rate decreased again but still increased compared with the control.

[0135] The phagocytosis activity of the water buffalo colostrum-derived bioactive peptides obtained by enzymolysis under the optimal enzymolysis conditions obtained by neutral protease in response surface optimization and their simulated gastrointestinal digestion products on macrophages RAW264.7 after 24 h is as shown in 10. In the figure, BWD is the water buffalo colostrum-derived bioactive peptide. The results showed that after intervention and culture with BWD, the cell phagocytosis rate was significantly increased compared with the control group. After BWD was simulated gastric digested, the cell phagocytosis rate decreased but still increased compared with the control. After the simulated gastric digestion products were further intestinal digested, the cell phagocytosis rate decreased again but still increased compared with the control.

[0136] The NO release amount of the water buffalo colostrum-derived bioactive peptides obtained by enzymolysis under the optimal enzymolysis conditions obtained by neutral protease in response surface optimization and their simulated gastrointestinal digestion products on macrophages RAW264.7 after 24 h is as shown in Figure 11As shown in the figure, BWD in the figure is the bioactive peptide from buffalo colostrum. The results showed that after being intervened and cultured with BWD, the NO release was significantly increased compared with the control group. After BWD was digested by simulated gastric juice, the NO release was significantly increased and even higher than that of the positive control. After the simulated gastric digestion products were further digested by intestinal juice, the cell phagocytosis rate decreased but still increased compared with the control. The NO detection method in this application: Detect according to the NO detection kit from Nanjing.

[0137] In vitro experiments, the proliferation, phagocytosis and NO release of macrophages RAW264.7 were used to evaluate the immunomodulatory effect of the bioactive peptide from buffalo colostrum. The results showed that although the cell proliferation rate and cell phagocytosis rate after digestion by simulated gastrointestinal fluids decreased, it may be because the produced immunomodulatory peptides were decomposed and lost their immunological activity after being treated with pepsin and trypsin, but they were all increased compared with the control group after digestion. The significant increase in NO release after simulated gastric digestion may be because more immunopeptides beneficial to promoting NO release were produced after pepsin hydrolysis, indicating that the bioactive peptide from buffalo colostrum has a certain stability after simulated gastrointestinal digestion.

[0138] 4. Antioxidant activity of simulated gastrointestinal digestion products

[0139] The results of the ABTS radical scavenging rate of the simulated gastrointestinal digestion products are shown in detail in Figure 12 ;

[0140] The ABTS radical scavenging rates of the bioactive peptide from buffalo colostrum and its simulated gastrointestinal digestion products obtained by enzymatic hydrolysis under the optimal enzymatic hydrolysis conditions obtained by neutral protease in response surface optimization are as Figure 12 shown in the figure. BWD in the figure is the bioactive peptide from buffalo colostrum. The results showed that the ABTS radical scavenging rate of BWD decreased after simulated gastric digestion, and the ABTS radical scavenging rate increased significantly and approached the level before digestion after the simulated gastric digestion products were further digested by intestinal juice.

[0141] The reducing power of the bioactive peptide from buffalo colostrum and its simulated gastrointestinal digestion products obtained by enzymatic hydrolysis under the optimal enzymatic hydrolysis conditions obtained by neutral protease in response surface optimization are as Figure 13 shown in the figure. BWD in the figure is the bioactive peptide from buffalo colostrum. The results showed that the cell reducing power of BWD decreased after simulated gastric digestion, and the cell reducing power recovered to the state before digestion after the simulated gastric digestion products were further digested by intestinal juice.

[0142] The antioxidant regulatory effect of buffalo colostrum-derived bioactive peptides was evaluated by measuring the ABTS radical scavenging rate and reducing power. The results showed that although the ABTS radical scavenging rate and reducing power decreased after digestion in simulated gastric juice, probably because the generated antioxidant peptides were decomposed by pepsin and lost their immunological activity, but after further digestion in simulated intestinal juice, the ABTS radical scavenging rate and reducing power were close to or even restored to the original state, indicating that buffalo colostrum-derived bioactive peptides had a certain stability after simulated gastrointestinal digestion.

[0143] Example 9:

[0144] From the experiment in Example 8, we obtained that the ABTS radical scavenging rate of buffalo colostrum-derived bioactive peptides decreased in gastric juice. Therefore, considering enteric coating treatment for the freeze-dried enzymatic hydrolysate and studying the ABTS radical scavenging rate of the product after coating treatment; among them, the method of coating treatment was: adding common excipients to the freeze-dried buffalo colostrum-derived bioactive peptides for tabletting, and using a coating machine to coat the tabletted buffalo colostrum-derived bioactive peptides, thus obtaining; among them, the coating material used for coating was: a mixture formed by ethylcellulose aqueous dispersion, talc powder, triethyl citrate and polyethylene glycol in a mass ratio of 10:2:1:0.1; the experimental results obtained were as follows:

[0145] Calculate the mass of the buffalo colostrum-derived bioactive peptides after coating treatment, weigh the buffalo colostrum-derived bioactive peptides (without coating treatment) with the same mass as the calculated amount as Control Group 1, weigh the whey protein powder (without coating treatment) with the same mass as the mass of the coated whey protein tablets as Control Group 2, and successively conduct gastrointestinal digestion experiments with simulated fluids. After the experiment, measure the ABTS radical scavenging rate of the digestion products, as shown in Table 9 specifically:

[0146] Table 9 ABTS radical scavenging rate of buffalo milk whey protein after coating treatment

[0147] Group ABTS radical scavenging rate Coated group 95.2％ Control group 1 87.8％ Control group 2 90.9％

[0148] As can be seen from Table 8, after enteric coating treatment of buffalo milk whey protein, its ABTS radical scavenging ability was better than that of Control Group 1 and Control Group 2. Thus, it shows that enteric coating treatment can significantly improve the stability of buffalo colostrum-derived bioactive peptides, with a higher ABTS radical scavenging rate and better immune function.

[0149] Therefore, enteric coating treatment should be carried out on the corresponding products when preparing the corresponding drugs or foods.

[0150] Example 10:

[0151] This example studied the effect of neutral protease on the hydrolysis reaction of whey protein under different enzyme addition amounts:

[0152] Among them, the enzymatic hydrolysis conditions of the BWD-1 experimental group were as follows: pH value 7, enzymatic hydrolysis time of neutral protease 3 h, and enzyme addition amount during enzymatic hydrolysis 3000 U / g; the enzymatic hydrolysis conditions of the BWD-2 experimental group were as follows: pH value 7, enzymatic hydrolysis time of neutral protease 3 h, and enzyme addition amount during enzymatic hydrolysis 4000 U / g; a negative control (100 ul of water) and a positive control (LPS: 100 ng / ml) were set up.

[0153] The results of the effects of different enzyme addition amount conditions on cell proliferation rate are as Figure 14 shown, and the results of the effects of different enzyme addition amount conditions on cell phagocytosis rate are as Figure 15 shown. Under different enzyme addition amount conditions, the proliferation activity of macrophages RAW264.7 treated with the enzymatic hydrolysis product after 24 h is as Figure 14 shown. The results showed that after intervention and culture with BWD1 or BWD2, the cell proliferation rate was significantly increased compared with the control group, and the proliferation rates of the BWD1 and BWD2 experimental groups were not much different. The phagocytosis activity of macrophages RAW264.7 treated with the enzymatic hydrolysis product after 24 h is as Figure 15 shown. The results showed that after intervention and culture with BWD1 or BWD2, the cell phagocytosis rate was increased compared with the control group, and the phagocytosis activity was significantly higher in the BWD1 group than in the BWD2 group after intervention and culture.

[0154] The results of the simulated artificial gastrointestinal experiment are as Figures 16 - 20 shown:

[0155] The results of the cell proliferation rate of the simulated gastrointestinal digestion products are shown in detail in Figure 16 ; it can be seen from Figure 16 that after intervention and culture with BWD1 or BWD2, the cell proliferation rate was significantly increased compared with the control group. After simulated gastric digestion with BWD1 and BWD1, the cell proliferation rate decreased but was still increased compared with the control. After the simulated gastric digestion product was further digested by the intestine, the cell proliferation rate would decrease again but was still increased compared with the control.

[0156] The phagocytosis activity of the product obtained after enzymatic hydrolysis and its simulated gastrointestinal digestion product on macrophages RAW264.7 after 24 h is as Figure 17 shown. It can be seen from the figure that after intervention and culture with BWD1 or BWD2, the cell phagocytosis rate was significantly increased compared with the control group. After simulated gastric digestion with BWD1 or BWD2, the cell phagocytosis rate decreased but was still increased compared with the control. After the simulated gastric digestion product was further digested by the intestine, the cell phagocytosis rate would decrease again but was still increased compared with the control.

[0157] The NO release amount of the product obtained after enzymatic hydrolysis and its simulated gastrointestinal digestion product on macrophages RAW264.7 after 24 h is as Figure 18As shown in the figure, it can be seen that after culturing with BWD intervention through BWD1 or BWD2, the NO release amount is significantly increased compared with the control group. After BWD is digested by simulated gastric juice, the NO release amount is significantly increased and even higher than that of the positive control. After the simulated gastric digestion products are further digested by intestinal juice, the cell phagocytosis rate will decrease but still increase compared with the control. In this application, the NO detection method: Detection is carried out according to the Nanjing NO detection kit.

[0158] In vitro experiments, the proliferation, phagocytosis and NO release of macrophages RAW264.7 were used to evaluate the immunomodulatory effect of buffalo colostrum-derived bioactive peptides. The results showed that although the cell proliferation rate and cell phagocytosis rate decreased after digestion by simulated gastrointestinal fluids, it may be because the generated immunomodulatory peptides were decomposed by pepsin and trypsin and lost their immunological activity. However, they were all increased compared with the control group after digestion. The significant increase in NO release after simulated gastric digestion may be because more immunopeptides beneficial to promoting NO release were generated after pepsin hydrolysis, indicating that buffalo colostrum-derived bioactive peptides have certain stability after simulated gastrointestinal digestion.

[0159] The ABTS radical scavenging rate results of simulated gastrointestinal digestion products are shown in Figure 19 ;

[0160] The ABTS radical scavenging rate of buffalo colostrum-derived bioactive peptides obtained after enzymatic hydrolysis and their simulated gastrointestinal digestion products is as Figure 19 shown. The results showed that the ABTS radical scavenging rate of BWD1 or BWD2 decreased after simulated gastric digestion. After the simulated gastric digestion products were further digested by intestinal juice, the ABTS radical scavenging rate increased significantly and approached the level before digestion.

[0161] The reducing power of buffalo colostrum-derived bioactive peptides obtained after enzymatic hydrolysis and their simulated gastrointestinal digestion products is as Figure 20 shown. In the figure, BWD is buffalo colostrum-derived bioactive peptide. The results showed that the cell reducing power of BWD1 or BWD2 decreased after simulated gastric digestion. After the simulated gastric digestion products were further digested by intestinal juice, the cell reducing power recovered to the state before digestion.

[0162] The antioxidant effect of buffalo colostrum-derived bioactive peptides was evaluated by measuring the ABTS radical scavenging rate and reducing power. The results showed that although the ABTS radical scavenging rate and reducing power decreased after digestion by simulated gastric juice, it may be because the generated antioxidant peptides were decomposed by pepsin and lost their antioxidant activity. However, after further digestion by simulated intestinal juice, the ABTS radical scavenging rate and reducing power were close to or even restored to the original state, further indicating that buffalo colostrum-derived bioactive peptides have certain stability after simulated gastrointestinal digestion.

[0163] In summary, the buffalo colostrum-derived bioactive peptides obtained by the enzymatic hydrolysis process of the present invention have the technical advantages of high ABTS free radical scavenging rate and good immune function. At the same time, the stability of the enzymatic hydrolysis product was further studied through gastrointestinal digestion experiments, providing a more comprehensive theoretical reference for the further research of whey protein.

[0164] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. Application of buffalo colostrum milk source active peptide in preparing drugs related to improving immunity, characterized in that, The preparation method of the bovine colostrum milk source active peptide is as follows: (1) Adjust the pH value of buffalo colostrum to (4.6 ± 0.2), and centrifuge at (4 ± 1) °C to remove the upper layer of fat and precipitate, obtaining a buffalo colostrum whey protein solution; (2) After adjusting the pH value of the buffalo colostrum whey protein solution obtained in step (1), preheat it for 8 - 10 minutes and then add neutral protease for constant temperature enzymatic hydrolysis to obtain an enzymatic hydrolysate; (3) Centrifuge the enzymatic hydrolysate obtained in step (2), take the supernatant, and adjust the pH to neutral to obtain the buffalo colostrum milk source active peptide; (4) Lyophilize the buffalo colostrum milk source active peptide obtained in step (3) and then perform enteric coating to obtain the drug; The enteric coating is prepared by mixing ethylcellulose aqueous dispersion, talcum powder, triethyl citrate and polyethylene glycol in a mass ratio of 10:2:1:0.1; In step (2), the pH value is 7.5, the enzymatic hydrolysis time of neutral protease is 2.0 h, and the enzyme addition amount during enzymatic hydrolysis is 6299 U / g; In step (1), the centrifugation conditions are centrifuging at 5000 ± 500 r / min for 20 min; In step (3), the centrifugation conditions are centrifuging at 8000 ± 500 r / min for 15 ± 2 min; In steps (1) to (3), the reagent used to adjust the pH value is 1M sodium hydroxide solution or 1M hydrochloric acid solution.

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