Preparation method of albumin active peptide and active peptide

Abstract

This invention relates to the field of deep processing technology for egg products, and particularly to a method for preparing albumin active peptides and the active polypeptides. The preparation method includes the following steps: S1, mixing egg white powder with water, keeping warm and allowing it to swell to obtain a liquid; S2, adding Bacillus licheniformis acidic protease to the liquid for hydrolysis to obtain a hydrolysate; S3, inactivating the enzyme in the hydrolysate, centrifuging to remove impurities, obtaining a supernatant, and then separating the supernatant through a membrane to retain the polypeptides, thus obtaining the albumin active peptides. These albumin active peptides have the property of protecting pepsin activity and can be used in food, pharmaceuticals, health products, or feed as a pepsin protectant; moreover, after enzymatic hydrolysis, the molecular weight of albumin is reduced, making it easier to absorb, and it can also be used as a nutritional supplement for postoperative recovery.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology for egg products, and in particular to a method for preparing albumin active peptides and active polypeptides. Background Technology

[0002] Pepsin, an aspartic acid protease, is widely used in numerous industries such as food processing, pharmaceuticals, and agriculture due to its excellent protein degradation capabilities. Applications include amino acid hydrolysis, peptide production, animal husbandry and feed processing, leather processing, juice clarification, and digestive aids. However, its poor thermal stability and narrow pH range limit its application. Specifically, pepsin exhibits poor stability and a short duration of action; it is easily inactivated at processing temperatures exceeding 60°C or pH > 5. To achieve good and sustained enzymatic hydrolysis, a high amount of active enzyme is required, increasing the cost of using pepsin. Therefore, food ingredients or additives that improve pepsin activity and stability have broad industrial value and market potential.

[0003] Studies on improving pepsin stability include: Bian Jinlin reported in "The Promoting Effect of Ya'an Tibetan Tea on Pepsin" that catechins in Ya'an Tibetan tea can enhance pepsin activity; the cited references in this article also reported that acidic substances such as organic acids, amino acids, and vitamin C can effectively enhance protease activity. Liu Xinze, in "The Effects of Adding Different Combinations of Acidifying Agents to Feed on Pepsin Activity," concluded that adding 0.1% (ammonium formate + essential oil) compound acidifying agent to the feed increased pepsin activity in broilers.

[0004] Research on processing egg white protein into bioactive peptides includes: Jin Lei's report in "Study on the Preparation of Bioactive Peptides from Egg White by Neutral Protease Hydrolysis" describes how using neutral protease 1398 to hydrolyze egg white protein yields oligopeptides with molecular weights primarily below 1350 Da. Chinese patent document CN107183308A discloses a method for obtaining egg white-derived bioactive peptides by alkaline protease hydrolysis of egg white protein powder. Chinese patent document CN110477185A discloses a method for processing antioxidant peptides from egg white protein by hydrolyzing it with the heat-stable protease THERMOASE PC10F.

[0005] In summary, no reports have yet been found of peptides that can protect pepsin or improve its thermal stability. Summary of the Invention

[0006] To address the shortcomings of the prior art mentioned in the background section, this invention provides a method for preparing albumin-active peptides, comprising the following steps:

[0007] S1. Mix egg white powder with water, keep warm and allow it to swell to obtain the liquid;

[0008] S2. Add Bacillus licheniformis acidic protease to the feed solution for hydrolysis to obtain hydrolysate;

[0009] S3. After inactivating the enzyme in the hydrolysate and centrifuging to remove impurities, the supernatant is obtained. The supernatant is then subjected to membrane separation to retain the polypeptide, thus obtaining the albumin active peptide.

[0010] Furthermore, in S1, the mass ratio of the egg white powder to water is 1:(10-15).

[0011] Furthermore, in S1, the temperature for heat preservation and expansion is 70-80℃, and the time is 60-120 minutes.

[0012] Furthermore, in S2, the amount of Bacillus licheniformis acidic protease added is calculated based on the protein content in the egg white powder, with an enzyme activity of 3000u-5000u per gram.

[0013] Furthermore, in S2, the pH of the feed solution is adjusted to 2.5-4.0, the hydrolysis temperature is 45-55℃, and the hydrolysis time is 60-120 min.

[0014] Furthermore, in step S3, the pH of the hydrolysate is first adjusted to 5.5-6.5, and then the hydrolysate is heated to 75-85°C to inactivate the enzyme.

[0015] Furthermore, in S3, the supernatant is separated by membrane separation to retain polypeptides with a molecular weight cutoff of 200-10000 Da, and then concentrated and dried to obtain powdered albumin active peptides.

[0016] Furthermore, the membrane separation method is as follows: the supernatant is ultrafiltered using a 10000 Da ultrafiltration membrane, and the resulting filtrate is then concentrated using a 200 Da nanofiltration membrane.

[0017] Preferably, the above drying method is freeze drying or spray drying.

[0018] The present invention also provides an active polypeptide, the components of which include the albumin active peptide obtained by the above preparation method.

[0019] Furthermore, the active polypeptide has the property of protecting pepsin activity.

[0020] Preferably, mixing albumin active peptides with pepsin with an activity of 4-11 kDa / g at a mass ratio of (2-4):1 can improve the thermal stability of pepsin.

[0021] This invention reveals that when albumin-active peptides are used as heat stabilizers in combination with pepsin, the initial pepsin activity should be less than 100 u / ml, and the mass ratio of the active peptide to pepsin should be (2-4):1. Within this range, the effect of improving pepsin stability is better. The principle may be that the 200-10000 Da albumin peptides of this invention, under certain concentrations and pepsin activity, can be further hydrolyzed to generate small peptides (molecular weight mainly below 3000 Da) that protect pepsin, thereby improving pepsin stability. When pepsin activity and albumin peptide concentration exceed a certain range, the amount of deprotective small peptide products generated by pepsin hydrolysis increases, thus weakening the protective effect of albumin peptides on pepsin.

[0022] Compared with the prior art, the method for preparing albumin active peptides and the active polypeptides provided by the present invention have the following beneficial effects:

[0023] (1) The processing technology of albumin active peptide provided by the present invention is simple, with few steps, and the obtained polypeptide has a high purity of more than 86%.

[0024] (2) Normally, pepsin is easily inactivated under conditions of pH > 5 or heating at 60-70℃. However, the albumin active peptide provided by this invention not only improves the thermal stability of pepsin and prolongs its active half-life by more than 50%, but also expands the pH activity range of pepsin. Specifically, after adding albumin active peptide to pepsin, its thermal stability and pH stability are significantly improved at pH 5-7 or when heated at 60-70℃ for 30-40 minutes.

[0025] (3) The albumin active peptide provided by the present invention can be further hydrolyzed into low molecular weight peptides after being mixed and dissolved with pepsin. More than 90% of the peptides are small molecules below 3000 Da. Since small molecules can be quickly absorbed by the human body, they can also be used as health products or nutritional products to restore physical strength. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 A graph showing the effect of different proportions of albumin-active peptides on porcine pepsin activity, provided by the present invention.

[0028] Figure 2The molecular weight distribution diagram of the sample provided for this invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] This invention provides an operational example of a method for preparing albumin-active peptides, the specific steps of which are as follows:

[0031] S1. Mix egg white powder and water at a mass ratio of 1:(10-15), keep warm at 70-80℃ for 60-120 minutes to obtain the liquid.

[0032] S2. Adjust the pH of the solution to 2.5-4.0, add Bacillus licheniformis acidic protease, and hydrolyze at 45-55℃ for 60-120 minutes to obtain hydrolysate; the amount of Bacillus licheniformis acidic protease added is based on the protein content in the egg white powder, with an enzyme activity of 3000u-5000u per gram.

[0033] S3. After adjusting the pH of the hydrolysate to 5.5-6.5, heat it to 75-85℃ to inactivate the enzyme for 10-15 minutes, centrifuge to remove insoluble matter, and obtain the supernatant. Then, ultrafilter the supernatant through a 10000Da ultrafiltration membrane, and concentrate the filtrate through a 200Da nanofiltration membrane to obtain albumin active peptides.

[0034] S4. Dry the albumin active peptides into powder to obtain albumin peptide powder.

[0035] The present invention provides the following embodiments and comparative examples:

[0036] Example 1

[0037] A method for preparing an albumin-active peptide is as follows:

[0038] S1. Take 100g of egg white powder (84% protein content), add 1000g of water and mix. Keep warm at 80℃ for 60 minutes to obtain the liquid.

[0039] S2. Adjust the pH of the solution to 2.6 with hydrochloric acid, then add 300 kDa of Bacillus licheniformis acidic protease (Shanghai Yuanju Biotechnology Co., Ltd.), and hydrolyze at 52℃ for 95 min to obtain hydrolysate;

[0040] S3. Adjust the pH of the hydrolysate to 6.0, then heat at 85℃ for 10 min to inactivate the enzyme; centrifuge to remove insoluble precipitate, and ultrafilter the supernatant first with a 10000 Da ultrafiltration membrane, and then concentrate the filtrate by nanofiltration with a 200 Da nanofiltration membrane to obtain albumin active peptide concentrate.

[0041] S4. The concentrate is freeze-dried to obtain albumin peptide powder with a molecular weight of 200-10000 Da and a polypeptide content of 86.8%.

[0042] This example demonstrates the effect of albumin-active peptides on porcine pepsin activity under different addition ratios:

[0043] Six portions of porcine pepsin with an activity of 4100 u / g were prepared, each weighing 1 gram. Five portions were mixed with 1 g (A1), 2 g (A2), 3 g (A3), 4 g (A4), and 5 g (A5) of albumin peptide powder prepared in Example 1, respectively. The remaining portion (A0) was not mixed with albumin peptide powder. The pure porcine pepsin powder A0 and the other five mixed enzyme powders were dissolved in 100 ml of water and 5 g of soy protein isolate to form sample solutions A0, A1, A2, A3, A4, and A5 (the initial activity of porcine pepsin was 41.0 u / ml). The pH was adjusted to 3.0 with hydrochloric acid, and then the solutions were heated at 70°C for different times. Samples were taken periodically and cooled in ice water to about 0°C for storage. The porcine pepsin activity of the sample solutions at each time point was then measured (using the national standard Folin-Ciocalteu method), and the half-life T was calculated. 1 / 2 The results are shown Figure 1 See Table 1.

[0044] Table 1. Activity (Tg) of porcine pepsin solutions with different albumin peptide addition ratios at 70℃ 1 / 2 With the rate of increase

[0045]

[0046] Note: Half-life T 1 / 2 =0.693 / k; k=(lnc1-lnc2) / t2-t1, t2-t1=-10, c1=41.

[0047] The above experiments show that the original enzyme (A0) without albumin peptide powder and A1 and A5 with albumin peptide powder added at ratios of 1:1 and 1:5, respectively, completely lost their activity after heating at 70℃ for 20 minutes. Although the enzyme half-life of A1 and A5 was prolonged relative to A0, the increase rate was less than 20%. When the ratio of albumin peptide powder to porcine pepsin was 2:1 (A2), 3:1 (A3), and 4:1 (A4), the enzyme half-life T of porcine pepsin was... 1 / 2Compared to the original enzyme A0, the activity increased by more than 150%. The enzymatic hydrolysates with addition ratios of 3:1 (A3) and 4:1 (A4) still exhibited pepsin activity after heating for more than 30 minutes, while the ratio of 2:1 (A2) showed activity after approximately 30 minutes. Therefore, the suitable addition ratio of this albumin peptide powder for porcine pepsin is (2-4):1. The principle may be that the 200-10000 Da albumin polypeptides obtained by hydrolysis with Bacillus licheniformis acidic protease can be further hydrolyzed under the action of porcine pepsin with a certain activity to generate polypeptides that protect porcine pepsin, with the small molecule peptides having a protective effect on porcine pepsin.

[0048] The decrease in protective activity against porcine pepsin when the albumin peptide ratio is 5:1 (A5) may be due to the following reasons: Upon contact with porcine pepsin, the added 200-10000 Da albumin peptides hydrolyze to release substances that protect pepsin, but may also produce substances detrimental to pepsin protection. When the albumin peptide ratio is appropriate, the protective peptides dominate, resulting in significant pepsin protection. However, when the albumin concentration is too high, the amount of hydrolysates detrimental to pepsin protection increases, weakening the protective effect against pepsin instability and potentially negating the protective effect of the peptides.

[0049] Example 2

[0050] A method for preparing an albumin-active peptide is as follows:

[0051] S1. Take 100g of egg white powder (84% egg white content), add 1500g of water and mix. Keep warm at 70℃ for 120 minutes to obtain the liquid.

[0052] S2. Adjust the pH of the solution to 3.5 with lactic acid, then add 420 kDa of Bacillus licheniformis acidic protease (Shanghai Yuanju Biotechnology Co., Ltd.), and hydrolyze at 45℃ for 120 min to obtain hydrolysate.

[0053] S3. Adjust the pH of the hydrolysate to 6.5, then heat at 75℃ for 15 min to inactivate the enzyme; centrifuge to remove insoluble precipitate, and ultrafilter the supernatant first with a 10000 Da ultrafiltration membrane, and then nanofilter the filtrate with a 200 Da nanofiltration membrane to obtain the concentrated albumin active peptide solution.

[0054] S4. The concentrate is spray-dried to obtain albumin peptide powder with a molecular weight of 200-10000 Da and a polypeptide content of 86.25%.

[0055] This example demonstrates the effect of albumin peptides on bovine pepsin activity.

[0056] Twenty-one portions of bovine pepsin with an activity of 9000 u / g were prepared, each containing 1 gram. Twelve portions were each mixed with 2 g of albumin peptide powder prepared in Example 2 (denoted as B1), while the remaining nine portions were not mixed with albumin peptide powder (denoted as B0). All 21 samples were dissolved in 100 ml of water (the initial bovine pepsin activity was 90 u / ml). Six portions of B1 and six portions of B0 were taken, and their pH values ​​were adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 respectively using hydrochloric acid and sodium carbonate. The samples were then heated simultaneously in a 60°C water bath for 30 minutes. After heating, the samples were stored at 0°C. The bovine pepsin activity and rate of change under each pH condition were then measured and calculated. The results are shown in Table 2.

[0057] Six additional B1 samples were taken and their pH values ​​were adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, respectively. Three B0 samples were taken and their pH values ​​were adjusted to 2.0, 3.0, and 4.0, respectively. All nine samples were placed in a 70°C water bath and heated simultaneously for 30 minutes. After the reaction, the samples were quickly placed in ice water and cooled to 0°C for storage. The bovine pepsin activity of the sample solutions was then measured under each pH condition, and the rate of change was calculated. The results are shown in Table 2.

[0058] Table 2. Changes in bovine pepsin activity at different pH values ​​with different albumin peptide addition ratios.

[0059]

[0060] Table 2 shows that, under the same temperature and pH conditions, the bovine pepsin activity in all B1 samples with added albumin peptides was significantly higher than that in the B0 samples without albumin peptides. After heating at 60℃ for 30 min, bovine pepsin remained active in B1 samples at pH 2-7, while it was inactivated in B0 samples at pH above 5. Furthermore, the bovine pepsin activity in B1 samples at pH 2-4 was on average 255.4% higher than that in B0. After heating at 70℃ for 30 min, the bovine pepsin activity in B0 samples at pH 2-4 was lost, while the bovine pepsin in B1 samples remained active at pH 2-6. Therefore, albumin peptides can not only improve the thermal stability of bovine pepsin but also enhance its pH stability and application range.

[0061] The albumin peptide powder prepared in Example 2 was diluted with water to a concentration of 20 mg / ml and designated as Group A. Separately, sample B1, which was hydrolyzed and enzyme-inactivated at pH 3 and heated to 60°C, was designated as Group B. The molecular weight distribution of Group A and Group B samples was detected using a UV detector and a G50 gel electrophoresis apparatus. The results are shown in [Figure 1]. Figure 2 And Table 3.

[0062]

[0063] from Figure 2 As shown in Table 3, before the albumin peptides prepared in Example 2 were mixed with bovine pepsin to form a liquid for hydrolysis, the mixed peptides were distributed in the range of less than 10,000 Da, with the largest elution peak appearing at 60.8 ml (corresponding to a molecular weight of 5210 Da). When bovine pepsin was added to sample B1 of the albumin peptides and hydrolyzed at 60°C for 30 minutes, the hydrolysate became a mixture of peptides mainly less than 5000 Da, with the main elution peak appearing at 91.5 ml (corresponding to a molecular weight of 1920 Da). The peptides were mainly small peptides below 3000 Da. Therefore, it is believed that after the albumin peptides of 200-10000 Da are further hydrolyzed into small peptides by bovine pepsin, a certain molecular weight of peptides has a protective effect on the structure of bovine pepsin, thus improving the thermal stability of bovine pepsin.

[0064] Example 3

[0065] A method for preparing an albumin-active peptide is as follows:

[0066] S1. Take 100g of egg white powder (84% protein content), add 1400g of water and mix. Keep warm at 75℃ for 90 minutes to obtain the liquid.

[0067] S2. Adjust the pH of the solution to 3.0 with hydrochloric acid, then add 336 kDa of Bacillus licheniformis acidic protease (Shanghai Yuanju Biotechnology Co., Ltd.), and hydrolyze at 50°C for 100 min to obtain hydrolysate;

[0068] S3. Adjust the pH of the hydrolysate to 5.8, then heat at 75℃ for 15 min to inactivate the enzyme; centrifuge to remove insoluble precipitate, and ultrafilter the supernatant first with a 10000 Da ultrafiltration membrane, and then nanofilter the filtrate with a 200 Da nanofiltration membrane to obtain the concentrated albumin active peptide solution.

[0069] S4. The concentrate is freeze-dried to obtain albumin peptide powder with a molecular weight of 200-10000 Da and a polypeptide content of 87.42%.

[0070] This example presents a comparative experiment on the effect of albumin peptides on porcine pepsin activity:

[0071] Eight samples of porcine pepsin with an activity of 7.3 kDa / g were taken, each containing 1 gram. Four samples were mixed with 4 g of albumin peptide powder prepared in Example 3 (denoted as C1), while the other four samples were not mixed with albumin peptide powder (denoted as C0). 100 ml of water was added to C0 and C1 respectively to dissolve them (the initial porcine pepsin activity of all eight samples was 73 kDa / ml). The pH was adjusted to 3.6. Then, 6 g of scallop protein powder was added to each sample. C0 and C1 were paired into four pairs and heated in water baths at 40℃, 50℃, 60℃, and 70℃ for 30 min respectively. After heating, they were quickly cooled to 0℃ in ice water and stored. The porcine pepsin activity of the samples at each time point was determined by the Folin-Ciocalteu method, and the peptide content was detected according to the method of GB / T 22492-2008. The results are shown in Table 4.

[0072] Table 4. Decrease in enzyme activity and polypeptide content of hydrolysate from scallop protein hydrolysate by pepsin at different temperatures.

[0073]

[0074] Table 4 shows that when porcine pepsin hydrolyzes scallop protein powder at 40-70℃, the peptide content of sample C1, which contains added albumin peptides, increases by 18-59% compared to sample C0, which does not contain albumin peptides. The higher the temperature, the greater the increase in peptide content, indicating that adding albumin peptides can improve the thermal stability and enzyme activity of porcine pepsin. After hydrolyzing scallop protein powder at 40-60℃ for 30 minutes, the pepsin activity of sample C1 decreases by 20-30% compared to sample C0 (ΔC). Therefore, it can be concluded that when albumin active peptides are mixed with porcine pepsin at a mass ratio of 2:1, the porcine pepsin in the solution is protected to a certain extent, and the ability to hydrolyze scallop protein is significantly improved.

[0075] Example 4

[0076] A method for preparing an albumin-active peptide is as follows:

[0077] S1. Take 100g of egg white powder (84% egg white content), add 1500g of water and mix. Keep warm at 75℃ for 90 minutes to obtain the liquid.

[0078] S2. Adjust the pH of the solution to 3.2 with hydrochloric acid, then add 252 kDa of Bacillus licheniformis acidic protease (Shanghai Yuanju Biotechnology Co., Ltd.), and hydrolyze at 50℃ for 120 min to obtain hydrolysate;

[0079] S3. Adjust the pH of the hydrolysate to 6.5, then heat at 80℃ for 12 min to inactivate the enzyme; centrifuge to remove insoluble precipitate, and ultrafilter the supernatant first with a 10000 Da ultrafiltration membrane, and then nanofilter the filtrate with a 200 Da nanofiltration membrane to obtain the concentrated albumin active peptide solution.

[0080] S4. The concentrate is freeze-dried to obtain albumin peptide powder with a molecular weight of 200-10000 Da and a polypeptide content of 87.20%.

[0081] Comparative Example 1

[0082] In step S2, the hydrolysis was performed using Aspergillus niger acidic protease 3.350 (a commercially available conventional enzyme preparation) with equal enzyme activity, replacing the Bacillus licheniformis acidic protease. All other steps were the same as in Example 4. The resulting albumin peptide powder had a polypeptide content of 84.18%.

[0083] Comparative Example 2

[0084] In step S2, *Bacillus licheniformis* acidic protease MSD (manufactured by Amano Corporation), with isoenzymatic activity, was used instead of MSD for hydrolysis. All other steps were the same as in Example 4. The resulting albumin peptide powder had a polypeptide content of 81.95%.

[0085] Comparative Example 3

[0086] In step S2, *Bacillus licheniformis* acidic protease 537 (a commercially available conventional enzyme preparation) with equal enzyme activity was used to replace the hydrolysis. All other steps were the same as in Example 4. The resulting albumin peptide powder had a polypeptide content of 86.52%.

[0087] Comparative Example 4

[0088] In step S2, the Bacillus licheniformis acidic protease was replaced with an isoenzymatically active Aspergillus honeysuckle protease (Amano Amano 3SD protease) for hydrolysis. The pH of the solution was 8.0, the hydrolysis temperature was 45°C, and the enzyme was inactivated at 95°C for 10 minutes after hydrolysis. All other treatments were the same as in Example 4. The resulting albumin peptide powder had a polypeptide content of 85.33%.

[0089] Comparative Example 5

[0090] In step S2, Bacillus licheniformis alkaline protease (a commercially available conventional enzyme preparation) with equal enzyme activity was used to replace Bacillus licheniformis acidic protease for hydrolysis. The pH of the solution was 10.0, the hydrolysis temperature was 60°C, and after hydrolysis, the enzyme was inactivated at 90°C for 10 minutes. All other treatments were the same as in Example 4. The resulting albumin peptide powder had a peptide content of 82.49%.

[0091] The albumin peptide powders obtained in Example 4 and Comparative Examples 1-5 were subjected to the following operations:

[0092] Two portions of porcine pepsin with an activity of 4100 u / g, each 1 gram, were added to 100 ml of water. One portion was dissolved with 3 grams of albumin peptide powder (denoted as D1), and the other portion was dissolved without albumin peptide powder (denoted as D0). 5 grams of defatted hemp seed protein powder were added to each of the two solutions to prepare sample solutions D0 and D1 respectively (the initial activity of porcine pepsin was 41 u / ml). The pH of the sample solution was adjusted to 3.2 with hydrochloric acid, and then heated at 60℃ for 10 minutes. The sample solutions were then quickly placed in ice water to cool to 0℃ and stored. The porcine pepsin activity C2 of the sample was then determined using the Folin method, and the half-life T was calculated. 1 / 2 The results are shown in Table 5.

[0093] Table 5. Activity of porcine pepsin in different solutions at 60℃ (T) 1 / 2

[0094]

[0095] Note: T 1 / 2 =0.693 / k; k=(l nC1-l nC2) / t2-t1; t2-t1=10, C1=41.

[0096] Table 5 shows that the enzyme activity T of each sample in Comparative Examples 1-5 1 / 2 The D0 values ​​were similar to those without albumin peptide, and significantly lower than those in Example 4, indicating that these comparative samples essentially did not improve the stability of porcine pepsin, while the enzyme activity T in Example 4... 1 / 2 T compared to D0 1 / 2 The extension was 279.68%, indicating that the albumin peptide provided in this embodiment of the invention can improve the stability of porcine pepsin at 60°C.

[0097] The active albumin peptide obtained in this invention was discovered incidentally during an antioxidant experiment, where it was found to protect pepsin activity. To verify whether other proteases hydrolyzed chicken egg white albumin had this activity, we conducted dozens of single-enzyme or complex protease hydrolysis experiments on albumin using the same method (not listed due to space limitations). Except for Bacillus licheniformis acidic protease, the hydrolysates of other enzymes did not significantly improve the stability of pepsin.

[0098] It should be noted that:

[0099] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. The albumin-active peptides of this application can be used to prepare solid dosage forms such as powders, tablets, and gels, and can also be dispersed in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0100] Unless otherwise specified, the raw materials used in this invention, such as porcine pepsin and bovine pepsin, are all existing commercially available products that can be purchased and obtained by those skilled in the art.

[0101] The reagents and instruments used in this embodiment do not specify the manufacturers or other information; they are all conventional products that can be purchased on the market.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing albumin-active peptides, characterized in that: Includes the following steps: S1. Mix egg white powder with water, keep warm and allow it to swell to obtain the liquid; S2. Add Bacillus licheniformis acidic protease to the feed solution for hydrolysis to obtain hydrolysate; S3. After inactivating the enzyme in the hydrolysate and centrifuging to remove impurities, the supernatant is obtained. The supernatant is then separated by membrane separation to retain the polypeptide, thus obtaining the albumin active peptide. In S2, the amount of Bacillus licheniformis acidic protease added is based on the protein content in the egg white powder, with an enzyme activity of 3000u-5000u per gram; In S2, the pH of the feed solution is adjusted to 2.5-4.0, the hydrolysis temperature is 45-55℃, and the hydrolysis time is 60-120 min; In S3, the supernatant is separated by membrane to retain polypeptides with a molecular weight cutoff of 200-10000 Da, and then concentrated and dried to obtain powdered albumin active peptides. The membrane separation method is as follows: the supernatant is ultrafiltered using a 10000 Da ultrafiltration membrane, and the filtrate is then concentrated using a 200 Da nanofiltration membrane.

2. The method for preparing albumin-active peptides according to claim 1, characterized in that: In S1, the mass ratio of egg white powder to water is 1:(10-15). The temperature for heat preservation and expansion is 70-80℃, and the time is 60-120 minutes.

3. The method for preparing albumin-active peptides according to claim 1, characterized in that: In step S3, the pH of the hydrolysate is first adjusted to 5.5-6.5, and then the hydrolysate is heated to 75-85℃ to inactivate the enzyme.

4. An active polypeptide, characterized in that: Its components include albumin active peptides prepared by the preparation method according to any one of claims 1-3.

5. The active polypeptide according to claim 4, characterized in that: The active polypeptide has the property of protecting pepsin activity.

Images