Egg white peptide capable of improving immunity activity as well as preparation method and application of egg white peptide

Abstract

The invention discloses an egg white peptide for improving immunity activity as well as a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The preparation method comprises the following steps: preparing egg white protein powder by taking egg white as a raw material and adopting an alkali extraction and acid precipitation method; alkaline protease immobilized by the alginate-based carrier is used for enzymolysis, and the immobilized enzyme can be repeatedly used and is high in efficiency and low in cost; and sequentially carrying out ultrafiltration separation and DEAE-52 cellulose ion exchange chromatography purification on the enzymatic hydrolysate, and precisely screening out high-activity components in combination with immunocompetence tracking. The molecular weight of the obtained egg white peptide is mainly distributed below 1 kDa, the purity reaches 92% or above, and the egg white peptide has definite good water solubility, is easy to absorb and has immunological enhancement activity. The egg white peptide can be used for preparing health-care food, formula food for special medical purposes and the like, high-value utilization of egg white resources is achieved, and the application prospect is wide.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to an egg white peptide that enhances immune activity, its preparation method, and its application. Background Technology

[0002] Immunity is a crucial defense mechanism for the human body to recognize and eliminate foreign substances, maintaining physiological homeostasis. Among numerous immune regulation strategies, food-derived immunomodulatory peptides demonstrate significant application potential due to their high activity, easy absorption, low allergenicity, and high safety. Food-derived peptides typically consist of 2-20 amino acid residues. They are not only digestible products of proteins but also important bioactive substances that can be absorbed into the circulatory system through the intestines in a manner similar to hormones or neurotransmitters. There, they interact with specific target organs or immune cells, thereby regulating the body's immune response.

[0003] Egg white, a natural treasure trove of high-quality protein, boasts a high protein content and a balanced amino acid composition, making it an ideal raw material for preparing bioactive peptides. Compared to whole animal eggs or other protein sources, egg white has the advantages of being low in fat and cholesterol, and is also widely available and inexpensive. Currently, the deep processing and utilization of egg white protein mainly focuses on its functional properties such as gelling and foaming properties, while research on the immunomodulatory peptides released through specific enzymatic hydrolysis techniques is still in a stage of continuous in-depth development.

[0004] Existing technologies have reported some research on egg white peptides, such as peptides with antioxidant, antibacterial (lysozyme), or antihypertensive (ACE inhibition) activities. However, research on egg white peptides specifically targeting the function of "enhancing immune activity" is still relatively limited and has some obvious limitations. First, many studies remain at the level of crude peptide mixtures, failing to clearly identify the core peptide sequences that play a key role, resulting in unstable product activity and unclear mechanisms of action. Second, conventional enzymatic hydrolysis processes (such as single enzymatic hydrolysis and imprecise control of the degree of hydrolysis) are often not highly targeted, potentially producing a large number of inactive or low-activity short peptides and free amino acids, which may mask or further degrade specific peptides with immune-enhancing potential, resulting in unsatisfactory functional activity of the final product. In addition, existing technologies lack systematic and in-depth mechanistic studies on the immunomodulatory effects of egg white peptides, such as their specific regulatory pathways on key immune cells such as macrophages, T lymphocytes, and B lymphocytes, and their precise effects on cytokine networks (such as interleukins, tumor necrosis factor, and interferons), which remain unclear. Summary of the Invention

[0005] In view of this, the purpose of this invention is to propose a method for preparing egg white peptides that is rationally processed, highly efficient, and produces products with clear immune-enhancing activity. This method uses egg white as raw material and obtains highly immune-active egg white peptides through specific extraction, enzymatic hydrolysis, and purification processes. Moreover, these egg white peptides can fully exert their physiological activity in the preparation of immune-enhancing products, while achieving efficient utilization of egg white resources.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention discloses a method for preparing egg white peptides that enhance immune activity, comprising the following steps:

[0008] (1) Egg white protein was extracted from the raw material using the alkaline extraction and acid precipitation method;

[0009] (2) Alkaline protease was immobilized on an alginate-based carrier to enzymatically hydrolyze the egg white protein obtained in step (1) to obtain the hydrolysate;

[0010] (3) The enzymatic hydrolysate obtained in step (2) is subjected to ultrafiltration and ion exchange chromatography to obtain the egg white peptide.

[0011] In some embodiments, the preparation method of egg white peptides specifically includes the following steps:

[0012] (1) Dissolve egg white in distilled water to make a solution, break it by sonication, adjust the pH to the preset value with NaOH solution, extract for a preset time, centrifuge to collect the supernatant; then adjust the pH of the supernatant to the isoelectric point with HCl, let it stand to precipitate, centrifuge again, collect the precipitate and adjust it to neutral, freeze dry to obtain egg white protein powder.

[0013] (2) Mix egg white protein powder with distilled water in proportion and heat to dissolve. Adjust the pH to the preset value with NaOH solution, add alkaline protease immobilized with alginate for enzymatic hydrolysis; then inactivate at high temperature, centrifuge to collect supernatant, adjust the pH to neutral with HCl, and freeze dry to obtain egg white peptide powder.

[0014] (3) Under the set conditions of membrane pressure, feed temperature and pH, ultrafiltration dialysis was performed at a preset reflux flow rate to separate different in vitro immune active components; the component with the highest activity was selected and freeze-dried to obtain egg white peptide freeze-dried powder.

[0015] (4) Further purification was carried out using DEAE-52 cellulose ion exchange chromatography: the egg white peptide lyophilized powder obtained in step (3) was prepared into a solution of a preset concentration with ultrapure water, filtered through a microfiltration membrane and loaded onto a equilibrated chromatography column; eluted with ultrapure water at a set flow rate, monitored and collected in separate tubes at a specified detection wavelength, and the in vitro immunological activity (including macrophage phagocytic capacity and cytokine secretion level) of the products in each tube was determined, thereby completing the preparation of egg white peptide.

[0016] Specifically, in step (2), the amount of alkaline protease immobilized on the alginate-based carrier added is 2000-3500 U / g (based on egg white protein content), the temperature of enzymatic hydrolysis is 35-50 ℃, and the time of enzymatic hydrolysis is 1-3 h.

[0017] Specifically, the preparation method of alkaline protease immobilized on the alginate-based carrier in step (2) includes:

[0018] Alkaline protease was uniformly dispersed in sodium alginate colloid under ice bath conditions to obtain a mixture. The mixture was then added dropwise to 0.1M CaCl2 solution at a rate of 22 mL / h through a 23G needle. Microspheres were formed under the condition that the needle was 5.5 cm away from the liquid surface and the cross-linking solution was stirred at 160 rpm. Finally, after solidification and washing, alkaline protease microspheres immobilized on an alginate-based carrier were obtained.

[0019] The amount of alkaline protease added is 45-55 mg of enzyme for every 1 g of sodium alginate.

[0020] In some embodiments, alkaline protease immobilized on an alginate-based carrier is prepared using an ionogel method. The specific steps are as follows:

[0021] (1) Dissolve 2.2 g sodium alginate powder in 100 mL phosphate buffer (0.05 M, pH 7.5), stir magnetically for 4.5 hours at room temperature to form a uniform and transparent colloidal solution, and then let it stand overnight to completely degas.

[0022] (2) Slowly add 110 mg of alkaline protease powder to 20 mL of the above sodium alginate colloid, and gently stir at 220 rpm for 35 minutes under ice bath conditions to make the enzyme evenly dispersed. This process helps to maintain enzyme activity and avoid foam generation.

[0023] (3) Using a syringe equipped with a 23G needle, the mixture was added dropwise to 200 mL of 0.1 M CaCl2 crosslinking solution at a constant rate of approximately 22 mL / h. The crosslinking process was carried out with slow stirring at 160 rpm, and the distance between the needle and the liquid surface was maintained at 5.5 cm to form microspheres with regular shape and uniform size. After the addition was completed, the crosslinking and curing was continued for 35 minutes to allow the microsphere structure to be completely stable.

[0024] (4) Collect microspheres with a sterile sieve, rinse thoroughly three times with pre-cooled ultrapure water to remove unfixed enzymes and residual CaCl2 on the surface, and store the resulting wet microspheres at 4°C for later use.

[0025] This invention uses an alginate-based carrier to immobilize alkaline protease because sodium alginate, as a natural polysaccharide, in Ca... 2+Under the influence of carboxyl groups, a three-dimensional "egg-box" shaped gel network is formed, thereby embedding and immobilizing the enzyme. This carrier has good biocompatibility, non-toxicity, and a porous structure, providing a stable microenvironment for the enzyme and facilitating the diffusion of substrates and products. The enzyme loading rate after immobilization can reach over 85%, its pH and temperature stability is significantly improved, and it can be reused more than 5 times, effectively reducing application costs.

[0026] The peptide mixtures produced by enzymatic hydrolysis exhibit significant differences in molecular weight and activity, with only components within specific molecular weight ranges possessing strong immunomodulatory activity. To accurately screen these highly active, uniformly molecularly derived components and eliminate the interference of impurities on subsequent activity evaluation and product development, the enzymatic hydrolysis products must be separated and purified. Therefore, establishing an efficient and simple separation and purification process, combined with immunomodulatory activity tracking for screening, is crucial for the industrial application of high-purity egg white peptides.

[0027] In some implementations, purification is performed using ultrafiltration and ion exchange chromatography.

[0028] Specifically, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1-8 kDa, and the operating conditions are: membrane pressure 1.8-4.2 Bar, temperature 28-38℃, membrane flux 3.5-7.5 LMH, and pH 6.5-9.5.

[0029] Preferably, the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1-8 kDa, and the operating conditions are: membrane pressure 1.8 Bar, temperature 28 ℃, membrane flux 5.5 LMH, and pH 7.5.

[0030] Ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa were used to fractionate the egg white protein hydrolysate by centrifugation. Four fractions with molecular weights <8 kDa, 4–8 kDa, 1–4 kDa, and <1 kDa were collected sequentially. Each fraction was freeze-dried and used for subsequent in vitro immunomodulatory activity assays. By comparing the activities of different molecular weight ranges, the range of peptides with optimal immunomodulatory effects can be identified, providing crucial information for subsequent fine purification.

[0031] The highly active components obtained above were further purified using DEAE-52 cellulose ion exchange chromatography. DEAE-52 cellulose, as a weak anion exchanger, can achieve efficient separation based on the difference in surface charge of peptide molecules, thereby removing impurity peptides and residual proteins to obtain egg white peptide components with a more concentrated molecular weight distribution and higher purity.

[0032] Secondly, this invention discloses an immunomodulatory egg white peptide, prepared by the aforementioned method. This egg white peptide has a molecular weight primarily below 1 kDa, a purity exceeding 92%, exhibits excellent immune-enhancing activity, and is highly water-soluble, easily absorbed, and safe.

[0033] Thirdly, the present invention discloses a health food product comprising the aforementioned immune-active egg white peptide and food-grade acceptable excipients.

[0034] Specifically, the immune-boosting health foods include, but are not limited to, oral liquids, compressed candies, freeze-dried powders, capsules, or meal replacement powders. The amount of egg white peptides added can be adjusted according to different product forms (usually 5%-20% of the total product mass) to fully exert their immune-regulating effects.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) High enzymatic hydrolysis efficiency and controllable cost: This invention uses an alginate-based carrier to immobilize alkaline protease. This immobilized enzyme provides a stable microenvironment for the enzyme molecules, which improves its enzymatic hydrolysis efficiency compared to unimmobilized enzymes; at the same time, the immobilized enzyme can be reused, significantly reducing the cost of enzyme preparations. In addition, the carrier raw materials are widely available and inexpensive, further controlling the overall production cost.

[0037] (2) Green economy of raw materials and high-value utilization: This invention uses egg white (including food processing by-products) as raw material. Not only is the raw material cost low and easy to obtain, which provides favorable conditions for industrialization, but it also realizes the high-value utilization of egg white resources and reduces the environmental pressure caused by waste discharge.

[0038] (3) The prepared egg white peptide products have the characteristics of good water solubility, easy absorption and high stability, and have been verified by in vitro and animal experiments to have clear immune-enhancing activity. The final product is easy to carry and convenient to consume, and can be mass-produced. It has broad application prospects in the fields of health food, special medical purpose formula food and dietary supplement. Detailed Implementation

[0039] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0040] In the following embodiments and comparative examples of this invention, alkaline protease immobilized on an alginate-based carrier was prepared using an ionogel method. The specific steps are as follows:

[0041] (1) Dissolve 2.2 g sodium alginate powder in 100 mL phosphate buffer (0.05 M, pH 7.5), stir magnetically for 4.5 hours at room temperature to form a uniform and transparent colloidal solution, and then let it stand overnight to completely degas.

[0042] (2) Slowly add 110 mg of alkaline protease powder to 20 mL of the above sodium alginate colloid, and gently stir at 220 rpm for 35 minutes under ice bath conditions to make the enzyme evenly dispersed. This process helps to maintain enzyme activity and avoid foam generation.

[0043] (3) Using a syringe equipped with a 23G needle, the mixture was added dropwise to 200 mL of 0.1 M CaCl2 crosslinking solution at a constant rate of approximately 22 mL / h. The crosslinking process was carried out with slow stirring at 160 rpm, and the distance between the needle and the liquid surface was maintained at 5.5 cm to form microspheres with regular shape and uniform size. After the addition was completed, the crosslinking and curing was continued for 35 minutes to allow the microsphere structure to be completely stable.

[0044] (4) Collect microspheres with a sterile sieve, rinse thoroughly three times with pre-cooled ultrapure water to remove unfixed enzymes and residual CaCl2 on the surface, and store the resulting wet microspheres at 4 °C for later use.

[0045] Example 1: A method for preparing egg white peptides that enhance immune activity

[0046] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0047] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 2200 U / g of alginate-based carrier to immobilize alkaline protease and hydrolyze at 38 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the hydrolysate;

[0048] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 2 Bar, feed temperature of 28 ℃, flow rate of 5.5 LMH and pH of 7.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0049] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0050] Example 2: A method for preparing egg white peptides that enhance immune activity

[0051] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0052] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 2500 U / g of alginate-based carrier to immobilize alkaline protease, and hydrolyze at 42 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the enzymatic hydrolysate;

[0053] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 3 Bar, feed temperature of 28 ℃, flow rate of 5.5 LMH and pH of 7.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0054] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0055] Example 3: A method for preparing egg white peptides that enhance immune activity

[0056] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0057] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 2800 U / g of alginate-based carrier to immobilize alkaline protease, and hydrolyze at 45 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the enzymatic hydrolysate;

[0058] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 4 Bar, feed temperature of 28 ℃, flow rate of 5.5 LMH and pH of 7.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0059] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0060] Example 4: A method for preparing egg white peptides that enhance immune activity

[0061] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0062] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 3000 U / g of alginate-based carrier to immobilize alkaline protease, and hydrolyze at 40 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the enzymatic hydrolysate;

[0063] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 1.8 Bar, feed temperature of 28 ℃, flow rate of 4 LMH and pH of 7.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0064] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0065] Example 5: A method for preparing egg white peptides that enhance immune activity

[0066] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0067] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 3200 U / g of alginate-based carrier to immobilize alkaline protease and hydrolyze at 48 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the hydrolysate;

[0068] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 1.8 Bar, feed temperature of 28 ℃, flow rate of 7 LMH and pH of 7.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0069] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0070] Example 6: A method for preparing egg white peptides that enhance immune activity

[0071] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0072] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 2600 U / g of alginate-based carrier to immobilize alkaline protease, and hydrolyze at 43 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the enzymatic hydrolysate;

[0073] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 1.8 Bar, feed temperature of 35 ℃, flow rate of 5.5 LMH and pH of 7.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0074] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0075] Example 7: A method for preparing egg white peptides that enhance immune activity

[0076] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0077] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 2400 U / g of alginate-based carrier to immobilize alkaline protease and hydrolyze at 40 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the hydrolysate;

[0078] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 1.8 Bar, feed temperature of 28 ℃, flow rate of 5.5 LMH and pH of 6.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0079] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0080] Example 8: A method for preparing egg white peptides that enhance immune activity

[0081] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0082] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 2900 U / g of alginate-based carrier to immobilize alkaline protease and hydrolyze at 46 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the hydrolysate;

[0083] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 1.8 Bar, feed temperature of 28 ℃, flow rate of 5.5 LMH and pH of 9.5. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0084] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0085] Example 9: A method for preparing egg white peptides that enhance immune activity

[0086] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0087] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 2700 U / g of alginate-based carrier to immobilize alkaline protease, and hydrolyze at 44 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the enzymatic hydrolysate;

[0088] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 3 Bar, feed temperature of 32 ℃, flow rate of 6 LMH and pH of 8. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0089] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0090] Comparative Example 1

[0091] Compared with Example 9, the difference is that ultrafiltration was omitted, but all other experimental steps are the same.

[0092] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0093] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, then add 2700 U / g of alginate-based carrier to immobilize alkaline protease, and hydrolyze at 44 °C for 2.5 h. Subsequently, inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the enzymatic hydrolysate;

[0094] (3) Subsequently, the cellulose was purified by DEAE-52 ion exchange chromatography: the enzymatic hydrolysate was loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0095] Comparative Example 2

[0096] The difference from Example 9 is that free alkaline protease was used for enzymatic hydrolysis, while the other experimental steps were the same.

[0097] (1) Dissolve egg white in distilled water, sonicate for 25 min, adjust pH to 10.5 with 0.1 mol / L NaOH solution, extract for 2.5 h, then centrifuge at 5500 r / min for 12 min to collect the supernatant. Adjust the pH of the supernatant to 4.5 (isoelectric point) with 0.1 mol / L HCl, let it stand for 2.5 h to precipitate, then centrifuge. The precipitate is neutralized and freeze-dried to obtain egg white protein powder;

[0098] (2) Dissolve egg white protein powder in distilled water at a ratio of 1:45 at 55 °C for 70 min. Adjust the pH to 10.5 with NaOH, add 2700 U / g of free alkaline protease, and hydrolyze at 44 °C for 2.5 h. Then inactivate the enzyme at 95 °C for 35 min, centrifuge and collect the supernatant to obtain the hydrolysate;

[0099] (3) The enzymatic hydrolysate obtained in step (2) was subjected to ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 8 kDa, 4 kDa, and 1 kDa under the conditions of membrane pressure of 3 Bar, feed temperature of 32 ℃, flow rate of 6 LMH and pH of 8. Components with different molecular weight ranges were collected. Components with molecular weight less than 1 kDa were identified as target egg white peptides. The target egg white peptides were freeze-dried to prepare egg white peptide freeze-dried powder for subsequent use.

[0100] (4) Then, a second purification was performed using DEAE-52 cellulose ion exchange chromatography: the active component egg white peptide lyophilized powder was prepared into a 55 mg / mL solution, loaded onto the equilibrated chromatography column, eluted with ultrapure water at a flow rate of 2.2 mL / min, and the active fraction was detected and collected at a wavelength of 252 nm.

[0101] Comparative Example 3

[0102] The difference from Example 5 is that alkaline protease was immobilized on a gelatin carrier, while the other experimental steps were the same.

[0103] The preparation steps of gelatin-supported immobilized alkaline protease include:

[0104] Weigh 3.0 g of gelatin powder and dissolve it in 100 mL of phosphate buffer (0.05 M, pH 7.5). Heat the solution in a 60 °C water bath and stir until completely dissolved to form a clear solution. After the solution cools to 35 °C, add 110 mg of alkaline protease powder and gently stir at 220 rpm for 30 minutes to ensure uniform dispersion of the enzyme.

[0105] Subsequently, the mixture was slowly added dropwise to 100 mL of isopropanol solution at 4 °C at a rate of 20 mL / h, while continuously stirring at 150 rpm to form gelatin microspheres. After the addition was complete, stirring was continued for 1 hour to solidify the mixture. Finally, the microspheres were collected using a sterile sieve and rinsed three times with pre-cooled ultrapure water to remove unfixed enzymes and residual isopropanol from the surface. The resulting microspheres were stored at 4 °C for later use.

[0106] Comparative Example 4

[0107] The difference from Example 5 is that alkaline protease was immobilized on a resin carrier, while the other experimental steps were the same.

[0108] The preparation steps of resin-supported immobilized alkaline protease include:

[0109] D380 macroporous anion exchange resin was selected, and after soaking in distilled water for 24 hours, it was pretreated by washing three times alternately with 0.5 mol / L HCl and 0.5 mol / L NaOH, and then equilibrated to neutral with 0.05 M phosphate buffer (PBS, pH 7.5).

[0110] Weigh 10.0 g of the treated resin and add it to 20 mL of PBS buffer (pH 7.5) containing 110 mg of alkaline protease. The mixture is shaken at 30 °C and 180 rpm for 4 hours to allow adsorption. After adsorption, wash the resin three times with PBS buffer to completely remove any unadsorbed enzyme. Store the resulting immobilized enzyme resin at 4 °C for later use.

[0111] Example 1: Determination of peptide yield and hydrolysis efficiency of egg white peptides

[0112] (1) Determination of peptide purity and proportion of small molecule peptides

[0113] Determination of peptide purity (reversed-phase high-performance liquid chromatography):

[0114] Solution preparation: Mobile phase A (0.1% TFA aqueous solution); Mobile phase B (0.1% TFA acetonitrile solution); Standard solution (accurately weigh BSA standard and prepare a 0.1 mg / mL solution with mobile phase A); Sample solution (accurately weigh each peptide sample, dilute to 1.0 mg / mL with mobile phase A, filter, and then test).

[0115] Chromatographic conditions: Column: C18 column (4.6 mm × 250 mm, 5 μm); Column temperature: 30 °C; Detection wavelength: 220 nm; Flow rate: 1.0 mL / min; Injection volume: 10 μL;

[0116] Gradient elution program: 0-5 min, 5% B; 5-30 min, 5% → 40% B; 30-35 min, 40% → 95% B; 35-40 min, 95% → 5% B.

[0117] Determination of the proportion of small molecule peptides (<1800 Da) (High performance size exclusion chromatography):

[0118] Solution preparation: Mobile phase (10 mM phosphate buffer, pH 6.8, containing 100 mM NaCl); Standard solution (accurately weigh a series of molecular weight standards and prepare a mixed standard working solution of 0.1 mg / mL with the mobile phase); Sample solution (accurately weigh each peptide sample, dilute to 1.0 mg / mL with the mobile phase, filter, and then test).

[0119] Chromatographic conditions: Column: TSKgel G2000SWXL (7.8 mm × 300 mm, 5 μm); Column temperature: 30 ℃; Detection wavelength: 220 nm; Mobile phase: phosphate buffer (isocratic elution); Flow rate: 0.5 mL / min; Injection volume: 20 μL.

[0120] All mobile phases and sample solutions were filtered through a 0.22 μm filter membrane and degassed by sonication before use. Three replicates of both samples and standards were prepared to ensure the reliability of the results.

[0121] (2) Determination of peptide yield

[0122] The supernatants after enzymatic hydrolysis of each example and comparative example were collected, and the total nitrogen content in the supernatant was determined by the Kjeldahl method. Based on the total nitrogen content in the egg white protein powder, the peptide yield was calculated according to the formula: Peptide yield (%) = (total nitrogen content in supernatant / total nitrogen content in egg white protein powder) × 100%.

[0123] (3) Hydrolysis efficiency determination

[0124] The trichloroacetic acid (TCA) precipitation method was used. 10 mL of the enzymatic hydrolysate was taken, 10 mL of 20% TCA solution was added, the mixture was shaken evenly and allowed to stand for 30 min. After centrifugation at 4000 r / min for 15 min, the protein content in the supernatant was determined (Lowry method). The hydrolysis efficiency was calculated based on the protein content in the egg white protein solution before enzymatic hydrolysis: Hydrolysis efficiency (%) = (TCA soluble protein content / total protein content before enzymatic hydrolysis) × 100%.

[0125] Table 1. Purity of egg white peptides and percentage of small molecule peptides in each group

[0126]

[0127] Table 2 Results of peptide yield and hydrolysis efficiency determination for each group

[0128]

[0129] Combining the data in Tables 1 and 2, it can be seen that the proportion of peptides <1000 Da in Comparative Example 1 (without ultrafiltration) (43.9%) is much lower than that in all examples, proving that ultrafiltration separation can effectively enrich small molecule peptides with higher immunogenic potential and is an indispensable purification step in this preparation method. Secondly, the peptide yield (78.5%) and hydrolysis efficiency (65.2%) of Example 5 are significantly higher than those of Comparative Examples 2-4, indicating that the alginate-based immobilized enzyme can more efficiently hydrolyze egg white protein into small molecule peptides, improving product yield and hydrolysis efficiency, further demonstrating the superiority of this immobilized enzyme.

[0130] Example 2: Enzymatic performance testing of alkaline protease immobilized on an alginate-based carrier.

[0131] (1) Determination of optimal pH

[0132] 0.05 M PBS buffer solutions with pH values ​​of 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 were prepared respectively. The enzyme preparations of Example 5 (alginate-based immobilized enzyme), Comparative Example 2 (free enzyme), Comparative Example 3 (gelatin-immobilized enzyme), and Comparative Example 4 (resin-immobilized enzyme) were added to the different pH buffer solutions and subjected to enzymatic hydrolysis at 40 °C (substrate was egg white protein solution). After 1 h of reaction, the enzyme activity was measured. The highest enzyme activity was taken as 100%, and the relative enzyme activity at different pH values ​​was calculated to determine the optimal pH and pH stability range (pH range with relative enzyme activity ≥80%).

[0133] (2) Thermal stability determination

[0134] Each of the above enzyme preparations was incubated at 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ for 1 h, respectively. Then, the enzyme activity was measured at its optimal pH and optimal temperature. The enzyme activity without incubation was taken as 100%, and the relative enzyme activity at different temperatures was calculated to evaluate the thermal stability.

[0135] (3) Determination of the number of times it can be reused

[0136] Each immobilized enzyme (free enzymes were not used in the reuse test) was used for protein hydrolysis of egg white. After each reaction, the enzyme preparation was collected by filtration, washed twice with PBS buffer, and used again for the next round of enzymatic hydrolysis. The operation was repeated until the enzyme activity dropped to less than 50% of the initial enzyme activity. The effective number of times the enzyme was reused (the number of times the relative enzyme activity was ≥50%) was recorded.

[0137] Table 3 Results of enzyme property determination for each enzyme preparation

[0138]

[0139] As shown in Table 3, the alginate-based immobilized enzyme of Example 5 exhibits a wider pH stability range (8.0-11.5) and superior thermal stability (78.3% relative enzyme activity after incubation at 60 °C for 1 h), and can be reused up to 8 times. This is significantly better than the other carrier-immobilized enzymes in Comparative Examples 3-4 (reusable 4-6 times) and the free enzyme in Comparative Example 2 (unreusable). This indicates that the alginate carrier can provide a more stable microenvironment for enzyme molecules, reducing the impact of external factors on enzyme activity. Furthermore, the recovery and reuse of the immobilized enzyme are more convenient, significantly reducing the cost of enzyme preparations.

[0140] Example 3: In vitro immunomodulatory activity assay

[0141] (1) Measurement of macrophage phagocytic capacity

[0142] Cell culture: Mouse peritoneal macrophages (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were adjusted to a density of 1×10⁶ cells / year using RPMI-1640 medium containing 10% fetal bovine serum. 6 Cells were seeded at a density of 100 μL / mL in 96-well plates. The plates were then incubated at 37 °C in a 5% CO2 incubator for 24 hours to allow cell adhesion.

[0143] Sample preparation: Discard the supernatant. Add 100 μL of culture medium containing different concentrations (10, 50, 100 μg / mL) of egg white peptide to the experimental groups, and add an equal volume of complete culture medium to the blank control group. Each group is divided into 3 replicates and cultured for 24 hours.

[0144] Phagocytosis reaction: Discard the supernatant, add 100 μL of 0.075% neutral red solution to each well, and continue incubation for 30 minutes. Then gently wash three times with PBS buffer to completely remove unphagocytosed neutral red.

[0145] Colorimetric analysis and measurement: Add 100 μL of cell lysis buffer (acetic acid:ethanol = 1:1, v / v) to each well and incubate at room temperature for 2 hours. After the cells have fully lysed and neutral red has been released, measure the absorbance of each well at a wavelength of 540 nm. The higher the absorbance value, the stronger the phagocytic capacity of the macrophages.

[0146] Table 4. Effects of egg white peptides on macrophage phagocytic capacity in each group

[0147]

[0148] The results are shown in Table 4. All examples exhibited significant immunomodulatory activity, showing a clear dose-dependent effect (i.e., the higher the concentration, the greater the absorbance value). This fully demonstrates that the egg white peptides prepared in each example of the present invention can effectively activate macrophages, enhance their phagocytic capacity, and have a clear in vitro immunomodulatory activity. At the three test concentrations, the absorbance value of Example 5 was the highest among all groups (0.468, 0.625, 0.753), indicating that the egg white peptides prepared there had the most prominent immunomodulatory effect. The phagocytic activity of Comparative Example 1 (without ultrafiltration) was significantly lower than that of most examples at all concentrations, especially Example 5. This indicates that the lack of an ultrafiltration step cannot effectively enrich highly active small molecule peptides, resulting in a significant reduction in the activity of the final product. The activities of Comparative Example 2 (using free enzyme), Comparative Example 3 (gelatin carrier), and Comparative Example 4 (resin carrier) were lower than those of all examples, and significantly lower than their corresponding immobilized enzyme groups (e.g., lower than Example 5). This shows that alginate carriers have unique advantages over other types of carriers in immobilizing alkaline proteases to produce highly active egg white peptides.

[0149] (2) Measurement of cytokine (TNF-α, IL-2) secretion levels (ELISA method)

[0150] Cell culture and processing: Following the cell culture and sample processing methods in "Assay of Macrophage Phagocytic Capacity", the concentration of mouse spleen lymphocytes was adjusted to 2 × 10⁻⁶. 6 The concentration of peptides was 500 μL / well in 24-well plates. Then, different concentrations of egg white peptide sample solutions were added, and the plates were incubated for another 48 hours.

[0151] Cytokine detection: After culture, the supernatant of each group was collected by centrifugation at 1500 r / min for 10 min. ELISA kits for TNF-α and IL-2 were used (according to the instructions). Standard curves were established using the kits, and the specific concentrations of each cytokine in the supernatant were calculated based on the absorbance values ​​of the samples.

[0152] Table 5. Effects of egg white peptides on the secretion of TNF-α and IL-2 by splenic lymphocytes in each group.

[0153]

[0154] The results are shown in Table 5. The egg white peptides prepared in this invention can significantly promote the secretion of immune cytokines, and the secretion levels of the two cytokines show a basically consistent trend. This proves that the egg white peptides prepared in this invention can effectively activate mouse splenic lymphocytes and promote the secretion of key immune cytokines TNF-α and IL-2, thereby enhancing immunity by regulating the body's immune response. Compared with Comparative Examples 1-4, Example 5 illustrates that the complete process with alginate immobilization enzymatic hydrolysis and ultrafiltration separation as the core is the key to obtaining high-purity egg white peptides with significant immune-enhancing activities (including activation of macrophages and promotion of lymphocyte cytokine secretion).

[0155] Example 4: In vivo experimental verification of the immunocompromised mouse model.

[0156] To further verify the immunomodulatory effect of egg white peptides on immunocompromised organisms in vivo, an immunocompromised mouse model induced by cyclophosphamide was established using the egg white peptides prepared in Example 5 as a sample, and in vivo immune function experiments were conducted:

[0157] 1. Laboratory animals and grouping

[0158] Sixty SPF-grade male Kunming mice were randomly divided into six groups (n=10): blank control group, model control group, positive control group (levamisole, 20 mg / kg) and low, medium and high dose groups of egg white peptide (50, 100 and 200 mg / kg).

[0159] 2. Model building and intervention

[0160] Except for the blank control group, all other groups were intraperitoneally injected with cyclophosphamide (80 mg / kg) for 2 consecutive days to establish an immunodeficiency model. After modeling, each dose group was administered the corresponding dose of egg white peptide solution by gavage, the positive control group was administered levamisole by gavage, and the blank and model control groups were administered the same volume of physiological saline by gavage, once daily for 14 days.

[0161] 3. Detection Indicators and Methods

[0162] The following parameters were measured 24 hours after the last gavage:

[0163] Immune organ index: Weigh the spleen and thymus, and calculate their ratio to body weight.

[0164] Peripheral blood lymphocyte transformation rate: The proliferative capacity of lymphocytes stimulated by ConA was detected by the MTT assay.

[0165] Serum hemolysin level: Antibody production capacity is measured by the hemolysin method to assess humoral immunity.

[0166] Phagocytic function of mononuclear macrophages: carbon clearance index (K) and phagocytic index (α) were calculated by carbon clearance assay.

[0167] As shown in Table 6, compared with the blank control group, the spleen index, thymus index, lymphocyte transformation rate, serum hemolysin level, carbon clearance index, and phagocytic index of mice in the model control group were significantly decreased (P<0.01), indicating that the immunocompromised mouse model was successfully established. Compared with the model control group, all immune indicators of mice in the low-, medium-, and high-dose egg white peptide groups and the positive control group were significantly increased (P<0.01), and in a dose-dependent manner. Among them, the spleen index (5.12±0.38%), thymus index (2.95±0.22%), lymphocyte transformation rate (62.1±4.0%), and serum hemolysin level (0.513±0.041) of the high-dose egg white peptide group were close to the level of the blank control group, and the carbon clearance index (0.038±0.004) and phagocytic index (6.15±0.48) were also significantly increased. The above results indicate that the egg white peptide prepared in this invention can effectively improve the immune function of cyclophosphamide-induced immunosuppressed mice. By increasing the immune organ index, promoting lymphocyte proliferation, enhancing humoral immune response and phagocytic capacity of mononuclear macrophages, it achieves in vivo immunomodulatory effects, further verifying its clear immune-enhancing activity.

[0168] Table 6. Effects of egg white peptides on immune function in immunocompromised mice.

[0169]

[0170] In summary, this invention utilizes egg white processing byproducts as raw materials and employs an efficient enzymatic hydrolysis and fractional purification technique using alginate-immobilized alkaline protease to prepare egg white peptides with definite immune-enhancing activity. This method combines resource recycling and cost advantages, is easily scaled up, and yields safe and effective products that can be widely applied in health foods, special medical foods, and other fields, demonstrating significant economic and social value.

[0171] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made using the present invention specification, or directly / indirectly applied to other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing egg white peptides that enhance immune activity, characterized in that, Includes the following steps: (1) Egg white protein was extracted from the raw material using the alkaline extraction and acid precipitation method; (2) Alkaline protease was immobilized on an alginate-based carrier to enzymatically hydrolyze the egg white protein obtained in step (1) to obtain the hydrolysate; (3) The enzymatic hydrolysate obtained in step (2) is subjected to ultrafiltration and ion exchange chromatography to obtain the egg white peptide.

2. The preparation method according to claim 1, characterized in that, In step (2), the amount of alkaline protease immobilized on the alginate-based carrier added is 2000-3500 U / g (based on egg white protein content), the enzymatic hydrolysis temperature is 35-50 ℃, and the enzymatic hydrolysis time is 1-3 h.

3. The preparation method according to claim 1 or 2, characterized in that, The preparation method of alkaline protease immobilized on the alginate-based carrier in step (2) includes: Alkaline protease was uniformly dispersed in sodium alginate colloid under ice bath conditions to obtain a mixture. The mixture was then added dropwise to 0.1M CaCl2 solution through a 23G needle at a rate of 22 mL / h. Microspheres were formed under the condition that the needle was 5.5 cm away from the liquid surface and the crosslinking solution was stirred at 160 rpm. Finally, after solidification and washing, alkaline protease microspheres immobilized on an alginate-based carrier were obtained.

4. The preparation method according to claim 3, characterized in that, The amount of alkaline protease added is 1 g of sodium alginate plus 45-55 mg of enzyme.

5. The preparation method according to claim 1, characterized in that, The ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 1-8 kDa, and the operating conditions are: membrane pressure 1.8-4.2 Bar, temperature 28-38℃, membrane flux 3.5-7.5 LMH, and pH 6.5-9.

5.

6. An immunologically active protein peptide, characterized in that, It is prepared by the method described in any one of claims 1-5.

7. A health food product, characterized in that, It comprises the immunologically active egg white peptide as described in claim 6 and food-acceptable excipients.

8. The health food according to claim 7, characterized in that, Its dosage forms include oral liquid, compressed candy, lyophilized powder, capsules, or meal replacement powder.

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