Method for preparing, enriching and separating various egg yolk bioactive peptides by enzymolysis of degreased egg yolk powder and application of egg yolk bioactive peptides

Abstract

The invention discloses a method for preparing, enriching and separating various egg yolk bioactive peptides through enzymolysis of defatted egg yolk powder and application of the egg yolk bioactive peptides, and belongs to the technical field of egg product processing and bioactive peptides. Egg yolk protein hydrolysate is prepared by adopting a three-step enzymolysis process, adsorption and gradient elution are performed on the egg yolk protein hydrolysate by adopting a macroporous resin chromatographic column, four components including water-1, water-2, 20% ethanol and 80% ethanol are obtained, the water-1 component is common egg yolk peptide, the water-1 component is enriched with osteogenic active peptide, the water-2 component is enriched with ethanol, and the 80% ethanol is enriched with ethanol. The 20% ethanol component enriches antioxidant peptide, and the 80% ethanol component enriches ACE inhibitory peptide. The functional activity of each bioactive peptide component is very strong, and the bioactive peptide component can be used as a high-end functional ingredient. According to the method disclosed by the invention, comprehensive utilization of the degreased yolk powder is realized, enrichment and separation of three active peptides are realized by using one raw material through a step-by-step enzymolysis-directional separation technology, the yield of the active peptides is greatly improved, and the economic benefits of enterprises are improved.

Description

Technical Field

[0001] The present invention relates to a method for preparing, enriching and separating multiple egg yolk bioactive peptides by enzymatic hydrolysis of defatted egg yolk powder and its application, belonging to the technical fields of egg product processing and bioactive peptides. Background Art

[0002] Defatted egg yolk powder is a by-product after extracting lecithin and egg yolk oil from egg yolk, rich in egg yolk protein, and its protein content can reach more than 75%. In recent years, studies have shown that egg yolk protein peptides have various biological activities such as antioxidant, metal chelation, blood pressure lowering, and promoting bone growth. They are ideal raw materials for developing functional foods and can be used in fields such as protecting cardiovascular health and promoting bone health.

[0003] However, existing research mostly focuses on the preparation of a single bioactive peptide. For example, in a method for preparing and efficiently enriching egg yolk phosphopeptide and its application recorded in the patent with publication number CN115537442A, the prepared egg yolk phosphopeptide has a high phosphorus element content, and has a high calcium ion chelating ability and the activity of promoting osteoblast proliferation, which is a kind of bioactive peptide. And by separating and purifying to obtain a highly active peptide, the yield is very low, resulting in low economic benefits. Especially, the proportion of by-products separated is large, but it is difficult to be utilized with high value, causing waste of resources. The above reasons limit the industrial production of egg yolk protein peptides. Summary of the Invention

[0004] Existing research mostly focuses on the preparation of a single bioactive peptide, and by separating and purifying to obtain a highly active peptide, the yield is very low, resulting in low economic benefits. Especially, the proportion of by-products separated is large, but it is difficult to be utilized with high value, causing waste of resources.

[0005] Therefore, this study proposes a method for preparing multiple egg yolk bioactive peptides by enzymatic hydrolysis of defatted egg yolk powder and enriching and separating them, synchronously obtaining three bioactive peptides (ACE inhibitory peptide, antioxidant peptide and osteogenic activity peptide, and the osteogenic activity peptide simultaneously has DPPH free radical scavenging ability and metal chelating ability), realizing the green comprehensive utilization of raw materials, greatly improving the economic benefits of enterprises, and promoting the industrial production of egg yolk protein peptide functional raw materials.

[0006] In order to improve the utilization rate of raw materials of defatted egg yolk powder, the present invention adopts a stepwise enzymatic hydrolysis - directional separation strategy: due to the strong hydrophobicity of defatted egg yolk powder, its solubility and dispersibility in aqueous solution are poor. First, a two-step enzymatic hydrolysis process is used to improve the yield of soluble protein, thereby enhancing the accessibility of protease cleavage sites; secondly, specific proteases are screened for directional enzymatic hydrolysis to simultaneously release ACE inhibitory peptides, antioxidant peptides, metal chelating peptides and osteogenic active peptides into the egg yolk protein hydrolysate. In order to obtain a bioactive peptide product with high activity, an industrial macroporous resin chromatography column is used for the enrichment and separation of bioactive peptides, and gradient elution is carried out according to the polarity differences of different bioactive peptides (the desorbing solution is an aqueous ethanol solution with different concentrations), realizing the efficient separation of three bioactive peptides.

[0007] At present, laboratory-level separation methods, such as solvent extraction, membrane filtration and chromatography, although they can extract a certain amount of bioactive peptides, problems such as high cost, low efficiency and poor selectivity limit their application in industrial production. The macroporous resin directional separation technology used in this study not only has renewable characteristics and physicochemical stability that can meet the requirements of continuous production, but also enriches and separates different bioactive peptides based on the structure-activity relationship of bioactive peptides. The N-terminal and C-terminal amino acids of ACE inhibitory peptides have hydrophobic characteristics, the phosphate groups, serine (Ser) and acidic amino acids (glutamic acid Glu, aspartic acid Asp) in metal chelating peptides (osteogenic active peptides) have strong hydrophilicity, and antioxidant peptides usually contain amino acids such as cysteine (Cys), methionine (Met), tyrosine (Tyr), histidine (His) that can provide hydrogen atoms and have certain hydrophilicity. Macroporous resin with certain hydrophobicity is selected to adsorb egg yolk protein peptides, and gradient desorption is carried out with an edible-grade aqueous ethanol solution commonly used in the food industry. According to the principle of "like dissolves like", the enrichment and directional separation of bioactive peptides with different polarities can be achieved. This technology has successfully realized the selective separation of multiple functional bioactive peptide components in the hydrolysate, providing an industrialized directional separation technical solution for the comprehensive preparation of multiple bioactive peptides from defatted egg yolk powder.

[0008] The present invention provides a method for preparing a multifunctional egg yolk protein hydrolysate, and the method comprises the following steps:

[0009] (1) Dissolution: After pulverizing defatted egg yolk powder, it is mixed with water at a mass-to-volume ratio of 1:10 to 1:7 to obtain a defatted egg yolk powder suspension;

[0010] (2) Ultrasonic pretreatment: The defatted egg yolk powder suspension obtained in step (1) is subjected to ultrasonic pretreatment;

[0011] (3) First enzymatic hydrolysis: Trypsin is added to the pretreated defatted egg yolk powder suspension obtained in step (2) for enzymatic hydrolysis;

[0012] (4) Second-step enzymatic hydrolysis: After the first-step enzymatic hydrolysis is completed, alkaline protease A is added for enzymatic hydrolysis;

[0013] (5) Third-step enzymatic hydrolysis: After the second-step enzymatic hydrolysis is completed, protease is added for enzymatic hydrolysis. The protease includes, but is not limited to: compound protease, ginger protease, flavor protease, neutral protease, papain;

[0014] (6) Preparation of multifunctional egg yolk protein hydrolysate: After the enzymatic hydrolysis is completed, the enzyme is inactivated, and the supernatant is obtained by centrifugation or filtration. The supernatant is concentrated and dried to obtain the product.

[0015] In one embodiment, in step (1), the defatted egg yolk powder is supercritical CO2 defatted egg yolk powder or ethanol-extracted defatted egg yolk powder; the conditions for ultrasonic treatment are: power is 250-350 W, reaction frequency is 15-25 kHz, and time is 5-20 min.

[0016] In one embodiment, the enzyme for the first-step enzymatic hydrolysis is trypsin, and the enzyme for the second-step enzymatic hydrolysis is alkaline protease A; the enzymatic hydrolysis conditions for adding trypsin are: enzyme-to-substrate ratio 1%-2% (E / S, w / w), enzymatic hydrolysis pH value 7.0-7.5, enzymatic hydrolysis temperature 37°C, enzymatic hydrolysis time 2-4 h; the enzymatic hydrolysis conditions for adding alkaline protease A are: enzyme-to-substrate ratio 2.0%-2.5% (E / S, w / w), enzymatic hydrolysis pH value 9.5-10.0, enzymatic hydrolysis temperature 48-52°C, enzymatic hydrolysis time 3-5 h;

[0017] In one embodiment, the enzyme for the third-step enzymatic hydrolysis is compound protease, and the enzymatic hydrolysis conditions are: enzyme-to-substrate ratio 0.6%-0.9% (E / S, w / w), enzymatic hydrolysis pH value 6.8-7.2, enzymatic hydrolysis temperature 48-52°C, enzymatic hydrolysis time 1-2 h;

[0018] In one embodiment, the enzyme for the third-step enzymatic hydrolysis is ginger protease, and the enzymatic hydrolysis conditions are: enzyme-to-substrate ratio 0.8%-1.0% (E / S, w / w), enzymatic hydrolysis pH value 6.8-7.2, enzymatic hydrolysis temperature 63-67°C, enzymatic hydrolysis time 0.5-1 h;

[0019] In one embodiment, the enzyme for the third-step enzymatic hydrolysis is flavor protease, and the enzymatic hydrolysis conditions are: enzyme-to-substrate ratio 0.8%-1.0% (E / S, w / w), enzymatic hydrolysis pH value 6.8-7.2, enzymatic hydrolysis temperature 53-57°C, enzymatic hydrolysis time 0.5-1 h;

[0020] In one embodiment, the enzyme for the third-step enzymatic hydrolysis is neutral protease, and the enzymatic hydrolysis conditions are: enzyme-to-substrate ratio 0.8%-1.0% (E / S, w / w), enzymatic hydrolysis pH value 6.8-7.2, enzymatic hydrolysis temperature 48-52°C, enzymatic hydrolysis time 0.5-1 h;

[0021] In one embodiment, the enzyme used in the third enzymatic hydrolysis is papain, and the enzymatic hydrolysis conditions are as follows: the enzyme-to-substrate ratio is 0.8%-1.0% (E / S, w / w), the enzymatic hydrolysis pH value is 6.8-7.2, the enzymatic hydrolysis temperature is 48-52°C, and the enzymatic hydrolysis time is 0.5-1 h;

[0022] The present invention also provides a multifunctional egg yolk protein hydrolysate prepared by the above preparation method.

[0023] The present invention also provides a method for simultaneously enriching and separating multiple bioactive peptides, and the method includes the following steps:

[0024] (1) Reconstitute the multifunctional egg yolk protein hydrolysate prepared by the above method or the above multifunctional egg yolk protein hydrolysate with water to obtain a sample; load and adsorb the obtained sample on a macroporous resin chromatography column; the macroporous resin includes but is not limited to DA201-C, XAD-1600, and XAD-16 macroporous resins;

[0025] (2) Gradient elution: Dynamically desorb the chromatography column with deionized water, 20% ethanol aqueous solution, 40% ethanol aqueous solution, and 80% ethanol aqueous solution in sequence, and collect each elution component according to the elution peak order, and name them water-1 component, water-2 component, 20% ethanol component, 40% ethanol component, and 80% ethanol component in sequence;

[0026] Or dynamically desorb the chromatography column with deionized water, 20% ethanol aqueous solution, and 80% ethanol aqueous solution in sequence, and collect each elution component according to the elution peak order, and name them water-1 component, water-2 component, 20% ethanol component, and 80% ethanol component in sequence;

[0027] (3) Concentrate and dry each elution component respectively to obtain each bioactive peptide component; the water-1 elution component obtained after drying is the osteogenic active peptide; the water-2 elution component obtained after drying is the ordinary egg yolk peptide; the 20% ethanol elution component obtained after drying is the antioxidant peptide; the 80% ethanol elution component obtained after drying is the ACE inhibitory peptide.

[0028] In one embodiment, in step (1), after reconstitution, the sample concentration is a solution of 150-250 mg / mL, and the loading pH value is 6.5-8.0.

[0029] In one embodiment, step (1) is to weigh 6 g of the multifunctional egg yolk protein hydrolysate, prepare it into a 150 mg / mL solution with deionized water, adjust the loading pH value to 7.5, and perform dynamic loading of the EYH solution with a constant flow pump.

[0030] In one embodiment, in step (2), the chromatography column is dynamically desorbed successively with deionized water, 20% aqueous ethanol solution, 40% aqueous ethanol solution, and 80% aqueous ethanol solution. The sample loading flow rate and the elution flow rate are both set to 3 mL / min, and the elution volume of each eluent (desorbent) is one column volume (180 mL); each elution component is collected in the order of elution peaks and named successively as water-1 (the first elution component eluted with water), water-2 (the second elution component eluted with water), Ethl-20% (i.e., the elution component collected with 20% aqueous ethanol solution), Ethl-40% (i.e., the elution component collected with 40% aqueous ethanol solution), and Ethl-80% (i.e., the elution component collected with 80% aqueous ethanol solution).

[0031] In one embodiment, in step (2), the chromatography column is dynamically desorbed successively with deionized water, 20% aqueous ethanol solution, and 80% aqueous ethanol solution. The sample loading flow rate and the elution flow rate are both set to 3 mL / min, and the elution volume of each eluent (desorbent) is one column volume (180 mL); each elution component is collected in the order of elution peaks and named successively as water-1 (the first elution component eluted with water), water-2 (the second elution component eluted with water), Ethl-20% (i.e., the elution component collected with 20% aqueous ethanol solution), and Ethl-80% (i.e., the elution component collected with 80% aqueous ethanol solution).

[0032] The present invention also provides a variety of bioactive peptides prepared by the above method, and the variety of bioactive peptides are osteogenic active peptides, common egg yolk peptides, antioxidant peptides, and ACE inhibitory peptides.

[0033] The present invention also provides the application of the osteogenic active peptide as a functional ingredient in the preparation of foods, functional foods, and / or health foods having the function of promoting bone growth or improving bone density.

[0034] The present invention also provides the application of the antioxidant peptide as a functional ingredient in the preparation of antioxidant foods, functional foods, and / or health foods.

[0035] The present invention also provides the application of the ACE inhibitory peptide as a functional ingredient in the preparation of health foods or functional foods for assisting in reducing hypertension.

[0036] In one embodiment, the health food also contains excipients; the dosage form of the health food can be solid powder, beverage, capsule, or tablet.

[0037] Beneficial effects

[0038] (1) Taking the three-step enzymatic hydrolysis method of trypsin, alkaline protease, and compound protease to enzymatically hydrolyze defatted egg yolk powder as an example, the soluble enzymatic hydrolysate yield of the egg yolk protein hydrolysate prepared with different enzymatic hydrolysis process parameters is between 76% and 80%, significantly improving the utilization rate of defatted egg yolk powder; on the other hand, the egg yolk protein hydrolysate has various biological activities, among which the ACE inhibition rate is between 80% and 83%, the ferrous ion chelation rate is between 62% and 65%, ABTS ·+ radical scavenging activity is between 445 and 466 μmol TE / g, the DPPH radical scavenging rate is between 53% and 54%, and it has a significant ability to promote osteoblast proliferation (134%);

[0039] (2) Using industrial-grade separation technology to directionally separate various bioactive peptides, with low cost, simple operation, and recyclability. Using a macroporous resin chromatography column, gradient elution is performed on the egg yolk protein hydrolysate (Egg Yolk Hydrolysate, EYH) (the eluent is water and ethanol aqueous solutions with different concentrations), and 4 components are obtained, namely: water-1 component, water-2 component, Ethl-20% component (the eluent is ethanol aqueous solution with a concentration of 20%), and Ethl-80% component (the eluent is ethanol aqueous solution with a concentration of 80%). Water-2 is used as a common egg yolk peptide; the water-1 component is enriched with osteogenic bioactive peptides (the ability to promote osteoblast proliferation is 178%), and at the same time has metal chelating ability (12.09 μg Fe 2+ / mg) and DPPH radical scavenging activity (IC 50 = 4.33 mg / mL or 9.16 μmol TE / g); the Ethl-20% component is enriched with antioxidant peptides (ABTS ·+ radical scavenging activity, TEAC value is 675.63 μmol TE / g; DPPH radical scavenging activity is 3.31 μmol TE / g); the Ethl-80% component is enriched with ACE inhibitory peptides (IC 50 = 0.26 mg / mL), a total of 3 bioactive peptide components. The functional activity of each bioactive peptide component is very strong and can be used as a high-end functional ingredient.

[0040] (3) Realized the comprehensive utilization of defatted egg yolk powder, a by-product of egg yolk processing. Using one raw material, the separation and enrichment of 3 bioactive peptides were achieved through a stepwise enzymatic hydrolysis-directional separation technology, greatly improving the yield of bioactive peptides (greater than 50%) and enhancing the economic benefits of the enterprise. Description of the Drawings

[0041] Figure 1 Adsorption and desorption amounts of six macroporous resins for EYH in Example 7; different uppercase or lowercase letters indicate significant differences (P < 0.05).

[0042] Figure 2 Adsorption kinetic curves of three macroporous adsorption resins for EYH in Example 7.

[0043] Figure 3 Elution curve of macroporous resin for EYH in Example 8.

[0044] Figure 4 Separation effects of macroporous resin under different sample loading amounts in Example 8. Among them, A is the sample recovery rate, B is the ACE inhibition rate, C is the ferrous ion chelation rate, D is the ABTS ·+ radical scavenging activity, and E is the DPPH radical scavenging rate.

[0045] Figure 5 Separation effects of macroporous resin under different sample loading pH values in Example 8. Among them, A is the sample recovery rate, B is the ACE inhibition rate, C is the ferrous ion chelation rate, D is the ABTS ·+ radical scavenging activity, and E is the DPPH radical scavenging rate; Different uppercase or lowercase letters indicate significant differences (P < 0.05).

[0046] Figure 6 pH values of each elution component solution under different sample loading pH values in Example 8.

[0047] Figure 7 Dispersion stabilities of each elution component solution under different sample loading pH values in Example 8. Among them, A is standing at room temperature for 24 h, and B is standing at room temperature for 48 h.

[0048] Figure 8 Sensory evaluations of each elution component under different sample loading pH values in Example 8. Among them, A is water-1, B is water-2, C is Ethl-20%, D is Ethl-40%, and E is Ethl-80%.

[0049] Figure 9 Separation effects of macroporous resin before and after omitting the 40% ethanol elution process in Example 8. Among them, A is the sum of the yields of Ethl-40% and Ethl-80% before omission and the yield of Ethl-80% after omission, and B is the ACE inhibition rates of Ethl-40% and Ethl-80% before omission and Ethl-80% after omission.

[0050] Figure 10 Separation effects of macroporous resin under different sample loading concentrations in Example 8. Among them, A is the sample recovery rate, B is the ferrous ion chelation rate, and C is the DPPH radical scavenging rate; Different uppercase or lowercase letters indicate significant differences (P < 0.05).

[0051] Figure 11Promoting proliferation activities of different bioactive peptides on MC3T3-E1 cells in Example 9; * indicates significant difference compared with the blank control group (P < 0.05); # indicates significant difference compared with EYH (P < 0.05).

[0052] Figure 12 This is the external view of the product of the present invention. Among them, A is EYH (multifunctional egg yolk protein hydrolysate), B is water-1 (osteogenic active peptide), C is water-2 (ordinary egg yolk peptide), D is Ethl-20% (antioxidant peptide), and E is Ethl-80% (ACE inhibitory peptide).

[0053] Figure 13 This is the technical roadmap of the present invention. Detailed implementation manners

[0054] The present invention provides a method for preparing, simultaneously separating and enriching multiple egg yolk bioactive peptides by enzymolysis of defatted egg yolk powder. To make the objectives, technical solutions and effects of the present invention clearer and more definite, the following will further elaborate on the present invention in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0055] The supercritical CO2 defatted egg yolk powder involved in the following embodiments is purchased from Yibin Yeyuan Phytochemical Co., Ltd., and the egg yolk powder is purchased from Bozhou Zhongyi Egg Industry Co., Ltd. Unless otherwise specified, w / v involved in the following embodiments represents g / mL. The data in the tables in the following embodiments are the averages after multiple repeated experiments. The 20% ethanol, 40% ethanol, and 80% ethanol involved in the following embodiments refer to: ethanol aqueous solutions with a volume fraction of 20%, ethanol aqueous solutions with a volume fraction of 40%, and ethanol aqueous solutions with a volume fraction of 80%. Ethl-20%, Ethl-40%, and Ethl-80% involved in the following embodiments are the 20% ethanol component, 40% ethanol component, and 80% ethanol, respectively.

[0056] The defatted egg yolk powder (referred to as ethanol-extracted defatted egg yolk powder), which is a by-product of extracting egg yolk oil and phospholipids with ethanol, can be purchased or prepared. The specific steps are as follows:

[0057] Mix 500 mL of absolute ethanol with 100 g of egg yolk powder (purchased from Bozhou Zhongyi Egg Industry Co., Ltd.), stir well to dissolve the egg yolk powder, use a magnetic stirrer to stir at 50 °C for 1.5 h, then filter to remove ethanol to obtain egg yolk powder. Repeat the above ethanol defatting step once, and finally place the filtered egg yolk powder in an oven at 50 °C for drying for 5 h, during which the egg yolk powder is stirred and mixed evenly every 1 h to obtain the ethanol-extracted defatted egg yolk powder, and store it at room temperature in a desiccator for later use;

[0058] Table 1: Composition of egg yolk powder

[0059]

[0060] The specific information of the protease involved in the following examples is shown in Table 2 below:

[0061] Table 2: Types of proteases used in each example of the present invention, their optimal pH values, temperatures and enzyme activities for enzymatic hydrolysis

[0062]

[0063]

[0064] The specific information of the macroporous resin involved in the following examples is shown in Table 3 below:

[0065] Table 3 Substrates, polarities, specific surface areas and pore diameters of different macroporous resin models

[0066]

[0067] The detection methods involved in the following examples are as follows:

[0068] Yield of soluble enzymatic hydrolysate of defatted egg yolk powder

[0069] Wash the precipitate generated by centrifugation during the preparation of the soluble enzymatic hydrolysate of defatted egg yolk powder into an evaporating dish of known mass with an appropriate amount of deionized water. Place the evaporating dish in an oven and dry it to a constant weight at a temperature of 105 °C. The yield of the soluble enzymatic hydrolysate is calculated according to the following formula:

[0070]

[0071] In the formula, m, m1, and m2 are the mass of supercritical CO2 defatted egg yolk powder, the mass of the precipitate and the evaporating dish after drying, and the mass of the evaporating dish, respectively.

[0072] Amount of inorganic salts (by-products) produced in the enzymatic hydrolysate of defatted egg yolk powder:

[0073] During the whole process of enzymatic hydrolysis, adjust the pH value of the egg yolk solution with 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid, record the volumes of the acid solution and the alkali solution used, and calculate the amount of by-product inorganic salts produced with Na + and Cl - The calculation formula is as follows:

[0074]

[0075] Determination of degree of hydrolysis

[0076] Dissolve 3.81 g of sodium tetraborate and 0.1 g of sodium dodecyl sulfate in 75 mL of deionized water; dissolve 80 mg of o-phthalaldehyde in 2 mL of absolute ethanol in the dark. After complete dissolution, transfer it to the above solution and rinse with deionized water. Subsequently, add 88 mg of dithiothreitol to the solution, transfer it to a 100 mL brown volumetric flask and make up to the mark to obtain the OPA reagent. Dilute the enzymolysis sample with deionized water to 1 mg / mL. Take 100 μL of the diluted sample and mix it with 1 mL of the OPA reagent, then shake immediately. After reacting for 2 min, take 200 μL of the mixture and transfer it to an enzyme-linked immunosorbent assay (ELISA) plate, and measure the absorbance at a wavelength of 340 nm. Using glutamic acid as the standard, measure the absorbance and plot the standard curve according to the same method: y = 0.2647x - 0.0064, R 2 = 0.9995. The blank group uses deionized water to replace the sample for the experiment. The calculation formula for the degree of hydrolysis is as follows:

[0077]

[0078] In the formula, N is the amino acid nitrogen concentration (mmol / L) in the hydrolyzed solution; N0 is the amino acid nitrogen concentration (mmol / L) in the hydrolyzed substrate solution; C is the substrate protein mass concentration (g / L), taking 0.75 g / L; H tot is the number of millimoles of peptide bonds per gram of raw material protein, taking 8 mmol / g.

[0079] Determination of ACE inhibition rate

[0080] Take 40 μL of the sample solution, sequentially add 50 μL of FAPGG (1 mM) and 10 μL of ACE (100 U / L), incubate at 37 °C for 5 min, and then incubate at 37 °C for 30 min. Record the change in absorbance at 340 nm, using HEPES buffer as the blank control. The sample concentration used in Examples 4 and 5 is 0.8 mg / mL, and the sample concentration used in Example 8 is 0.5 mg / mL. The calculation formula for the ACE inhibition rate is as follows:

[0081]

[0082] In the formula: ΔA0 is the decrease in absorbance of the blank group; ΔA x is the decrease in absorbance of the sample group.

[0083] Determination of ferrous ion chelation rate

[0084] The Ferrozine method was used to determine the ferrous ion chelating activity of the hydrolysate. Take 800 μL of the sample solution (1 mg / mL), add 25 μL of 2 mM FeCl2 and 50 μL of 5 mM Ferrozine, mix well, and after reacting for 10 min at room temperature, measure the absorbance at a wavelength of 562 nm, with deionized water as the blank control. The calculation formula for the ferrous ion chelating rate is as follows:

[0085]

[0086] Where: A0 is the absorbance of the blank group; A1 is the absorbance of the sample group.

[0087] ABTS ·+ Determination of free radical scavenging activity

[0088] Mix 7.0 mM ABTS solution and 2.45 mM potassium persulfate, and place it in the dark at room temperature for 12 - 16 h to obtain the ABTS ·+ stock solution. Dilute the ABTS ·+ stock solution with PBS buffer (0.01 M, pH = 7.4) until its absorbance value at 734 nm is 0.70 ± 0.02 to obtain the ABTS ·+ working solution. Take 20 μL of the sample solution, add 180 μL of the ABTS ·+ working solution, shake well and react in the dark for 6 min, and measure its absorbance value at 734 nm, using distilled water instead of the sample as the blank control. Make a standard curve of the ABTS ·+ free radical scavenging rate (y, %) against the Trolox concentration (x, μM): y = 0.2921x + 2.485, R 2 = 0.9973, and express the ABTS ·+ free radical scavenging activity of the sample in terms of TEAC value (μmol TE / g). The sample concentration used in Examples 4 and 5 is 0.5 mg / mL, and the sample concentration used in Example 8 is 0.3 mg / mL. The calculation formula for the ABTS ·+ free radical scavenging rate is as follows:

[0089]

[0090] Where: A0 is the absorbance of the blank group; A1 is the absorbance of the sample group.

[0091] Determination of DPPH free radical scavenging rate

[0092] Take 500 μL of the sample solution, add 200 μL of 0.02% DPPH solution, mix well, and react in the dark for 30 min. Measure the absorbance at a wavelength of 517 nm. Use deionized water instead of the sample as the blank control, and use absolute ethanol instead of the DPPH solution as the sample blank control. Make a standard curve of the DPPH radical scavenging rate (y, %) against the Trolox concentration (x, μM): y = 1.2673x - 0.4583, R 2 = 0.9993. The sample concentration used in Examples 4 and 5 is 8 mg / mL, and the sample concentration used in Example 8 is 4 mg / mL. The calculation formula for the DPPH radical scavenging rate is as follows:

[0093]

[0094] In the formula: A c is the absorbance of the blank control group; A b is the absorbance of the sample blank group; A s is the absorbance of the sample group.

[0095] Detection of the peptide content of the sample, calculation of the adsorption amount and desorption amount:

[0096] The Folin-phenol method was used to detect the peptide content. Take 0.5 mL of the sample solution (containing about 20 - 250 μg / mL of polypeptide or protein), add 2.5 mL of Folin-phenol reagent A, mix well, and let it stand at room temperature for 10 min. Then add 0.25 mL of Folin-phenol reagent B, shake immediately, and let it stand at room temperature for 30 min. Measure the absorbance at 750 nm, using 0.5 mL of water instead of the sample as the blank control. The calculation formulas for the adsorption amount and desorption amount are as follows:

[0097]

[0098] In the formula: C0 is the peptide content (mg / mL) in the supernatant when t = 0; C t is the peptide content (mg / mL) in the supernatant at the adsorption time t; C1 is the peptide content (mg / mL) in the desorption solution; V i is the volume (mL) of the EYH solution added; V1 is the volume (mL) of the desorption solution; W is the dry weight (g) of the macroporous resin; q t is the adsorption amount (mg / g) of the macroporous resin for the peptide at the adsorption time t; q1 is the desorption amount (mg / g) of the macroporous resin for the peptide.

[0099] Calculation of the yield of each elution fraction and the sample recovery rate

[0100] The calculation formulas are as follows:

[0101]

[0102] Determination of the pH value of each elution component solution:

[0103] According to the determination method of GB / T 9724-2007, use standard buffer solutions with pH values of 7.00, 4.01, and 9.21 to calibrate the pH meter in sequence, so that the measured slope is within the range of 90%-100%. Mix the freeze-dried powder of each elution component separated at the loading pH values of 6.5, 7.5, and 8.0 with deionized water in a ratio of 1:20 (w / v) evenly, and measure the pH value of the sample solution.

[0104] Determination of the dispersion stability of each elution component:

[0105] Mix the freeze-dried powder of each elution component separated at the loading pH values of 6.5, 7.5, and 8.0 with deionized water in a ratio of 1:20 (w / v) evenly. Respectively pipette 2.5 mL of the solution and transfer it into 15 5-mL transparent centrifuge tubes, place them on a transparent centrifuge tube rack, and store them at room temperature for 24-48 h. Observe the appearance of the solution, and take pictures every three as a group for recording.

[0106] Sensory evaluation of each elution component:

[0107] Prepare the freeze-dried powder of each elution component separated at the loading pH values of 6.5, 7.5, and 8.0 and 5% (w / v) solution, and let it stand at room temperature for 10 min. According to GB / T 16291.1-2012, select 8 trained sensory evaluators to conduct sensory evaluation on the samples. The sensory score is the comprehensive score of the sample appearance, odor, taste, tissue state, and overall acceptance (abbreviation "overall"). The total score uses a 5-point system, and the higher the score, the better the sensory quality. During the whole evaluation process, ensure the quietness and comfort of the evaluation environment to avoid interference from external factors. At the same time, keep the evaluation process standardized and standardized to ensure the objectivity and accuracy of the evaluation results. The sensory scoring criteria are shown in Table 4, corresponding to 1-5 points from left to right.

[0108] Table 4: Sensory scoring criteria for bioactive peptides

[0109]

[0110] Detection of the proliferation activity of promoting MC3T3-E1 cells

[0111] (1) Preparation of cell culture medium:

[0112] MC3T3-E1 complete culture medium: α-MEM basal medium + 10% FBS + 1% P / S;

[0113] MC3T3-E1 differentiation medium: α-MEM basal medium + 10% FBS + 1% P / S + 10 mM β-glycerophosphate + 50 μg / mL ascorbic acid.

[0114] (2) Cell resuscitation and subculture:

[0115] Take the cryopreservation tube of MC3T3-E1 cells and place it in a 37°C water bath. Shake it quickly to thaw. After there is no crystal, transfer the cells to a centrifuge tube containing complete medium, centrifuge at 1000 rpm for 3 min, and discard the supernatant. Resuspend the precipitate with complete medium, pipette evenly, inoculate it into a culture dish, add an appropriate amount of medium, and culture it in an incubator at 37°C and 5% CO2.

[0116] When the cell density reaches 80%-90%, discard the supernatant, and rinse with calcium- and magnesium-free PBS 1-2 times. Add 1-2 mL of 0.25% trypsin (containing EDTA) digestion solution, digest at 37°C for 1-3 min. Observe under a microscope until the cells become round and detached, then add complete medium to terminate digestion, and pipette evenly. Centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend with complete medium, and aliquot and place it in a cell culture plate according to the ratio for continuous culture.

[0117] (3) Determination of cell proliferation activity:

[0118] Determined by the MTT method. The cells in the cell culture plate are cultured with complete medium until the density reaches 80%, and then replaced with complete medium containing different samples (100 μg / mL) and cultured for 24 h. Then, each well is replaced with complete medium containing 0.5 mg / mL concentration of MTT and cultured in a cell incubator for 4 h. Discard the medium, and add 150 μL of DMSO to dissolve the purple crystals.

[0119] The absorbance value at 490 nm is measured by an enzyme-linked immunosorbent assay (ELISA) reader. The survival rate of the control group is set at 100%, and calculated according to the formula:

[0120]

[0121] Determination of molecular weight distribution

[0122] The molecular weight distribution is determined by size exclusion chromatography. Dissolve the sample in ultrapure water to prepare a 2 mg / mL solution, filter it with a 0.22 μm aqueous filter membrane, take 10 μL of the sample filtrate, manually inject it into a TSK gel G2000SWXL chromatographic column (7.8×300 mm, 5 μm) for detection. The mobile phase is 45% acetonitrile (containing 0.1% TFA), the flow rate is 0.5 mL / min, the detection wavelength is 220 nm, and the elution time is 30 min.

[0123] Determination of amino acid composition

[0124] Determined by the pre-column derivatization method of HPLC-phenyl isothiocyanate.

[0125] Total hydrolysis of the sample: Weigh 8 mg of the sample and dissolve it in 8 mL of 6 M hydrochloric acid. Hydrolyze it in an oven at 110 °C for 24 h. After the total hydrolysis of the sample is completed, take 1 mL of the hydrolyzate into an evaporating dish and place it in an oven at 42 °C to allow the hydrochloric acid to volatilize and dry completely. Then, redissolve it with 1 mL of 0.1 M hydrochloric acid for standby.

[0126] Pre-column derivatization: Mix 100 μL of 0.1 M phenyl isothiocyanate, 100 μL of 1 M triethylamine, and 200 μL of the redissolved sample in a centrifuge tube. After vortexing and mixing evenly, let it stand in the dark for 1 h. Then, add 400 μL of n-hexane, vortex for 1 min, and let it stand for 10 min. Pipette the lower clear liquid and filter it through a 0.22 μm organic filter membrane.

[0127] HPLC analysis: Take 10 μL of the sample filtrate and manually inject it into an Agilent Advance Bio AAA chromatographic column (4.6×100 mm, 2.7 μm); Mobile phase A is 10 mM disodium hydrogen phosphate and 10 mM sodium tetraborate, and the pH value is adjusted to 8.2 with hydrochloric acid; Mobile phase B is methanol-acetonitrile-water (45:45:10); The elution flow rate is 1.0 mL / min; The detection wavelength is 254 nm, and the elution gradient is: 0 - 0.35 min, 2% B; 0.35 - 6.9 min, 2% - 22% B; 6.9 - 13.4 min, 22% - 56% B; 13.4 - 13.5 min, 56% - 100% B; 13.5 - 15.7 min, 100% B; 15.7 - 15.8 min, 100% - 2% B; 15.8 - 18 min, 2% B.

[0128] Example 1: Preparation process of defatted egg yolk powder hydrolysate and hydrolysis effect under different pretreatment processes

[0129] The specific steps are as follows:

[0130] 1. Method for preparing defatted egg yolk powder hydrolysate with alkaline protease A:

[0131] (1) Dissolution: Grind the supercritical CO2 defatted egg yolk powder, pass it through a 60-mesh sieve, and mix it with deionized water in a ratio of 1:10 (w / v) and stir evenly to obtain a defatted egg yolk powder suspension;

[0132] (2) Hydrolysis: Adjust the pH value of the defatted egg yolk powder suspension to 10.0 with 1 mol / L sodium hydroxide solution, add alkaline protease A (see Table 5), the enzyme-to-substrate (supercritical CO2 defatted egg yolk powder) ratio (E / S) is 1% (w / w), the hydrolysis temperature is 50 °C, and the hydrolysis time is 3 h;

[0133] (3) Inactivation of enzymes: After the enzymatic hydrolysis is completed, treat with boiling water for 10 min. After cooling to room temperature, adjust the pH value of the defatted egg yolk powder suspension to 7.0 with 1 mol / L hydrochloric acid;

[0134] (4) Centrifugation: Place the feed liquid in a 50 mL centrifuge tube and centrifuge in a centrifuge at a speed of 6000 rpm for 10 min. The supernatant is the soluble enzymatic hydrolysate of defatted egg yolk powder; collect the precipitate in the lower layer for standby.

[0135] 2. Preparation method of the microwave pretreatment - alkaline protease A group:

[0136] Different from the alkaline protease A group, a microwave pretreatment process is added before the (2) enzymatic hydrolysis step. The mode is P50 and the microwave time is 10 min.

[0137] 3. Preparation method of the ultrasonic pretreatment - alkaline protease A group:

[0138] Different from the alkaline protease A group, an ultrasonic pretreatment process is added before the (2) enzymatic hydrolysis step. The power is 300 W, the reaction frequency is 20 kHz, the ultrasonic mode is on for 3 s and off for 3 s, and the ultrasonic time is 10 min.

[0139] 4. Method for preparing enzymatic hydrolysate of defatted egg yolk powder with alkaline protease B

[0140] Different from the alkaline protease A group, the enzymatic hydrolysis step in step (2) is adjusted as follows: Adjust the pH value of the defatted egg yolk powder suspension to 8.5 with 1 mol / L sodium hydroxide solution, add alkaline protease B (see Table 5), the mass of the enzyme is 1% of the mass of the substrate (supercritical CO2 defatted egg yolk powder), the enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis time is 3 h;

[0141] 5. Preparation method of the homogenization pretreatment - alkaline protease B group:

[0142] Different from the alkaline protease B group, a homogenization pretreatment process is added before the (2) enzymatic hydrolysis step. The rotation speed is 10000 rpm and the homogenization time is 5 min.

[0143] Determine the yield of the soluble enzymatic hydrolysate of defatted egg yolk powder obtained by different treatment methods. The results are shown in Table 5:

[0144] Table 5: Yield of soluble enzymatic hydrolysate of defatted egg yolk powder under different preparation methods

[0145] Preparation method of defatted egg yolk powder enzymolysate Yield of soluble enzymolysate (%) Alkaline protease group A 28.4 Microwave pretreatment - Alkaline protease group A 28.7 Ultrasonic pretreatment - Alkaline protease group A 42.0 Alkaline protease group B 32.2 Homogenization pretreatment - Alkaline protease group B 25.2

[0146] The results showed that: after ultrasonic pretreatment, the yield of soluble hydrolysate under the treatment of alkaline protease A was greatly improved. Ultrasonic treatment could change the structure of defatted egg yolk powder, expose more potential cleavage sites, improve the accessibility of protease cleavage sites, and thus increase the yield of soluble hydrolysate. However, microwave pretreatment and homogenization pretreatment had no obvious improvement effect on the yield. Therefore, it was determined to add ultrasonic pretreatment process before enzymolysis.

[0147] Example 2: Yield and inorganic salt generation amount of defatted egg yolk powder hydrolysate under different enzymolysis methods

[0148] 1. Preparation method of defatted egg yolk powder hydrolysate under single protease enzymolysis method

[0149] (1) Dissolution: After grinding supercritical CO2 defatted egg yolk powder, it was sieved through a 60-mesh sieve and mixed with deionized water at a ratio of 1:10 or 1:3 (w / v) and stirred evenly to obtain a defatted egg yolk powder suspension;

[0150] (2) Ultrasonic pretreatment: The defatted egg yolk powder suspension was subjected to ultrasonic pretreatment with a power of 300 W, a reaction frequency of 20 kHz, a supermode of 3 s on and 3 s off, and an ultrasonic time of 10 min;

[0151] (3) Respectively use the enzymes shown in Table 2 to enzymolyze the ultrasonic pretreated defatted egg yolk powder suspension obtained in step (2):

[0152] Use 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid to adjust the pH value of the defatted egg yolk powder suspension to the optimal pH value of each enzyme respectively, add the corresponding protease and stir evenly, and then dispense it into Erlenmeyer flasks. The samples were placed in a water bath oscillator (adjusted to the optimal temperature of the enzyme) for enzymolysis. The optimal pH value and optimal temperature of various proteases are shown in Table 2, and the enzyme-to-substrate (supercritical CO2 defatted egg yolk powder) ratio (E / S) and enzymolysis time are shown in Table 6;

[0153] (4) Inactivation of enzyme: After the enzymolysis was completed, it was treated with boiling water for 10 min. After cooling to room temperature, the pH value of the defatted egg yolk powder suspension was adjusted to 7.0 with 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid;

[0154] (5) Centrifugation: The liquid material was placed in a 50 mL centrifuge tube and centrifuged in a centrifuge at a speed of 6000 rpm for 10 min. The supernatant was the soluble hydrolysate of defatted egg yolk powder; the lower layer precipitate was collected for standby.

[0155] 2. Preparation method of defatted egg yolk powder hydrolysate under combined enzymolysis method

[0156] Different from single-enzyme enzymolysis, the enzymolysis step in step (3) was adjusted as follows:

[0157] Adjust the pH value of the defatted egg yolk powder suspension to 8.5 with 1 mol / L sodium hydroxide solution, and simultaneously add alkaline protease B and Alcalase. The ratio of each enzyme to the substrate (supercritical CO2 defatted egg yolk powder) (E / S) is 0.5% (w / w). The enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis time is 3 h.

[0158] 3. Preparation method of enzymolysis product of defatted egg yolk powder by two-step enzymolysis

[0159] Different from single-enzyme enzymolysis, adjust the enzymolysis step in step (1) as follows: After grinding the supercritical CO2 defatted egg yolk powder, pass it through a 60-mesh sieve, and mix it with deionized water at a ratio of 1:10 or 1:7 or 1:5 (w / v) and stir evenly to obtain a defatted egg yolk powder suspension;

[0160] After adjusting the enzymolysis step in step (3), add a second-step enzymolysis treatment. The optimal pH value and optimal temperature of various proteases are shown in Table 2, and the ratio of each enzyme to the substrate (supercritical CO2 defatted egg yolk powder) (E / S), addition sequence, and enzymolysis time are shown in Table 6; in the two-step enzymolysis, for example: alkaline protease A, 3 h + trypsin, 3 h, which means: in the first-step enzymolysis, alkaline protease A is added, and after 3 h of enzymolysis, in the second-step enzymolysis, trypsin is added and enzymolyzed for 3 h;

[0161] Measure the yields of soluble enzymolysis products of defatted egg yolk powder and the production amounts of by-product inorganic salts obtained by the three methods respectively. The results are shown in Table 6.

[0162] Table 6: Yields of soluble enzymolysis products of defatted egg yolk powder and production amounts of inorganic salts (by-products) under different enzymolysis methods

[0163]

[0164]

[0165] The results show that:

[0166] (1) Using single protease enzymolysis, the yields of soluble enzymolysis products under single enzymolysis of alkaline protease A and B are between 34% and 57%; when alkaline protease A is enzymolyzed alone, a material-liquid ratio of 1:10 is better than 1:3; as the enzymolysis time of alkaline protease A extends, the yield of soluble enzymolysis products gradually increases; when the enzymolysis time of alkaline protease A is 6 h, as E / S increases, the yield also increases, and the highest yield is 57%.

[0167] (2) Using the combined enzyme method (alkaline protease B + Alcalase, 3 h), the yield does not increase significantly under the condition of constant total enzyme addition amount.

[0168] (3) Using the two-step enzyme method, the yields are all above 40%:

[0169] The yield is significantly improved by the enzymatic hydrolysis method with pepsin first and then pancreatin compared with the single-enzymatic hydrolysis with pepsin or pancreatin alone, and the yield can reach 61%.

[0170] For different combinations of alkaline protease A and pancreatin, the yields are all above 40%. Moreover, the order of enzymatic hydrolysis has a significant impact on the yield of soluble hydrolysates. The yield of soluble hydrolysates is higher when pancreatin is added first and then alkaline protease A than when alkaline protease A is added first and then pancreatin. The yield is as high as 73.6%, which is significantly better than single-enzymatic hydrolysis, and the production of by-product inorganic salts is relatively low (2.09%).

[0171] The yields of soluble hydrolysates of defatted egg yolk powder under the combination of three material-liquid ratios and pancreatin first and then alkaline protease A are between 41.3% and 73.6%. With the increase of the material-liquid ratio, the yield of soluble hydrolysates shows a downward trend. Under the same enzyme dosage and hydrolysis time, when the material-liquid ratio is 1:10 (w / v), the yield of soluble hydrolysates is the highest, which is 52.6%. When the material-liquid ratio increases to 1:7, the yield is 47.5%. Considering that enzymatic hydrolysis can save energy in the later processing under the condition of high material-liquid ratio, and the yield of soluble solids can be increased by increasing the enzyme dosage (enzyme-to-substrate ratio), the material-liquid ratio of the enzymatic hydrolysis reaction can be controlled between 1:10 and 1:7 (w / v).

[0172] Example 3: Enzymatic hydrolysis effect of defatted egg yolk powder under different enzymatic hydrolysis process parameters (two-step enzymatic hydrolysis method)

[0173] The specific steps are as follows:

[0174] (1) Dissolution: After grinding the supercritical CO2 defatted egg yolk powder, it is sieved through a 60-mesh sieve and mixed with deionized water at a ratio of 1:10 (w / v) and stirred evenly to obtain a defatted egg yolk powder suspension;

[0175] (2) Ultrasonic pretreatment: The defatted egg yolk powder suspension is subjected to ultrasonic pretreatment with a power of 300 W, a reaction frequency of 20 kHz, an ultrasonic mode of 3 s on and 3 s off, and an ultrasonic time of 10 min;

[0176] (3) First-step enzymatic hydrolysis: Pancreatin is used for single enzymatic hydrolysis, and the enzymatic hydrolysis temperature is fixed at 37 °C. The ratio of enzyme to substrate (supercritical CO2 defatted egg yolk powder) (E / S), enzymatic hydrolysis time, and enzymatic hydrolysis pH value are shown in Table 7;

[0177] (4) Second-step enzymatic hydrolysis: After the first-step enzymatic hydrolysis is completed, alkaline protease A is added for single enzymatic hydrolysis, and the enzymatic hydrolysis temperature, the ratio of enzyme to substrate (supercritical CO2 defatted egg yolk powder) (E / S), enzymatic hydrolysis time, and enzymatic hydrolysis pH value are shown in Table 7;

[0178] (5) Inactivation of enzymes: After the enzymatic hydrolysis is completed, the mixture is treated with boiling water for 10 min. After cooling to room temperature, the pH value of the material liquid is adjusted to 7.0 with 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid;

[0179] (6) Centrifugation: The material liquid is placed in a 50 mL centrifuge tube and centrifuged in a centrifuge at a speed of 6000 rpm for 10 min. The supernatant is the soluble enzymatic hydrolysate of defatted egg yolk powder; the lower precipitate is collected for later use.

[0180] The yield of the soluble enzymatic hydrolysate of defatted egg yolk powder after the test was measured, and the results are shown in Table 7:

[0181] Table 7: Enzymatic hydrolysis effects under different enzymatic hydrolysis process parameters (two-step enzymatic hydrolysis method)

[0182]

[0183] The results showed that when the enzyme dosage of trypsin (calculated according to the enzyme-to-substrate ratio (E / S)) was 1%-2%, the enzymatic hydrolysis time of trypsin was 2-4 h, the pH value of trypsin enzymatic hydrolysis was 7.0-7.5, the enzyme dosage of alkaline protease A (calculated according to the enzyme-to-substrate ratio (E / S)) was 2.0%-2.5%, the enzymatic hydrolysis time of alkaline protease A was 3-5 h, the pH value of alkaline protease A enzymatic hydrolysis was 9.5-10.0, and the enzymatic hydrolysis temperature of alkaline protease A was in the range of 48-52 °C, a relatively high yield (66.4%-74.3%) was obtained, and the production of by-product inorganic salts was less (less than 2.5%).

[0184] Example 4: Preparation of multifunctional egg yolk protein enzymatic hydrolysate from defatted egg yolk powder under different enzymatic hydrolysis process parameters (three-step enzymatic hydrolysis method)

[0185] The specific steps are as follows:

[0186] (1) Dissolution: After grinding the supercritical CO2 defatted egg yolk powder, it is passed through a 60-mesh sieve and mixed with deionized water in a ratio of 1:10 (w / v) and stirred evenly to obtain a defatted egg yolk powder suspension;

[0187] (2) Ultrasonic pretreatment: The defatted egg yolk powder suspension is subjected to ultrasonic pretreatment with a power of 300 W, a reaction frequency of 20 kHz, an ultrasonic mode of 3 s on and 3 s off, and an ultrasonic time of 10 min;

[0188] (3) First-step enzymatic hydrolysis: The pH value of the defatted egg yolk powder suspension is adjusted to 7.5 with 1 mol / L sodium hydroxide solution, and trypsin is added. The ratio of enzyme to substrate (supercritical CO2 defatted egg yolk powder) (E / S) is 1.0%, the enzymatic hydrolysis temperature is 37 °C, and the enzymatic hydrolysis time is 2 h;

[0189] (4) Second enzymatic hydrolysis: After the first enzymatic hydrolysis, adjust the pH value of the material liquid to 9.9 with 1 mol / L sodium hydroxide solution, add alkaline protease A, the ratio of enzyme to substrate (supercritical CO2 degreased egg yolk powder) (E / S) is 2.5%, the enzymatic hydrolysis temperature is 50 °C, and the enzymatic hydrolysis time is 4 h;

[0190] (5) Third enzymatic hydrolysis: Adjust the pH value of the material liquid to the optimal pH value of each enzyme with 1 mol / L sodium hydroxide solution or 1 mol / L hydrochloric acid, add protease, the ratio of enzyme to substrate (supercritical CO2 degreased egg yolk powder) (E / S) is 1%, the enzymatic hydrolysis temperature is the optimal temperature of each enzyme, and the enzymatic hydrolysis times are 0.5 h and 1 h respectively. The types of proteases used are shown in Table 8, and the optimal enzymatic hydrolysis conditions of various proteases are shown in Table 2;

[0191] (6) Enzyme inactivation: After the enzymatic hydrolysis is completed, treat it with boiling water for 10 min. After cooling to room temperature, adjust the pH value of the material liquid to 7.0 with 1 mol / L hydrochloric acid;

[0192] (7) Centrifugation: Place the material liquid in a 50 mL centrifuge tube, centrifuge it in a centrifuge at a speed of 6000 rpm for 10 min, vacuum concentrate and freeze-dry the supernatant, which is the multi-functional egg yolk protein hydrolysate for the determination of biological activity; collect the lower precipitate for the determination of the yield of soluble hydrolysate.

[0193] Detect the yields, degrees of hydrolysis, ACE inhibition rates, ferrous ion chelation rates, ABTS ·+ radical scavenging activities and DPPH radical scavenging rates of the soluble hydrolysates of degreased egg yolk powder prepared under different three-step enzymatic hydrolysis process parameters respectively. The results are shown in Table 8:

[0194] Table 8: Yields, degrees of hydrolysis and biological activities of soluble hydrolysates of degreased egg yolk powder after three-step enzymatic hydrolysis

[0195]

[0196] The results show that: After three-step enzymatic hydrolysis, the yields of the soluble hydrolysates of degreased egg yolk powder are between 71.0% and 82.8%.

[0197] When the third enzyme hydrolyzes for 0.5 h, the degrees of hydrolysis of the products of enzymes F, J, and P increase to about 25%, and the degrees of hydrolysis of the products of other enzymes are all stable at about 20%. When the enzymatic hydrolysis time continues to extend to 1 h, the degrees of hydrolysis of most hydrolysates show an increasing trend. The degree of hydrolysis of enzyme J is the highest, and the degrees of hydrolysis of the products of enzymes M, Z, T, and D remain stable, and the degree of hydrolysis is basically saturated.

[0198] The hydrolysates of P enzyme (0.5 h and 1 h) and Z enzyme (0.5 h) had the highest ACE inhibitory activity, followed by the hydrolysates of F enzyme (1 h), Z enzyme (1 h), B enzyme (0.5 h), G enzyme (1 h) and M enzyme (1 h), all of which were significantly higher than those of the control group (P < 0.05).

[0199] The hydrolysates of A enzyme (0.5 h and 1 h), G enzyme (0.5 h and 1 h) and D enzyme (1 h) had the highest ferrous ion chelating activity, followed by the hydrolysate of D enzyme (0.5 h), which was significantly higher than that of other groups (P < 0.05).

[0200] The type of the third enzyme had less influence on the antioxidant activity. The hydrolysate of G enzyme (1 h) had the highest ABTS ·+ radical scavenging activity, followed by T enzyme (0.5 h and 1 h) and A enzyme (1 h), which were significantly higher than those of the control group (P < 0.05). The DPPH radical scavenging rates of the hydrolysates of G enzyme (1 h), M enzyme (0.5 h) and Z enzyme (0.5 h) were the highest, followed by M enzyme (1 h), D enzyme (0.5 h), Z enzyme (1 h) and G enzyme (0.5 h), and all were significantly increased compared with the control group (P < 0.05).

[0201] Based on the above determination results of the degree of hydrolysis and biological activities, the preferred enzymes for the third-step hydrolysis were P enzyme (compound protease), G enzyme (ginger protease), F enzyme (flavor protease), Z enzyme (neutral protease) and M enzyme (papain). The degrees of hydrolysis of the 1-h hydrolysates of these five proteases were between 21.50% and 26.14%, and all had high ACE inhibition rates (62.59% - 77.33%), ferrous ion chelation rates (48.81% - 62.36%) and antioxidant activities (ABTS ·+ radical scavenging activity of 445.95 - 489.09 μmol TE / g and DPPH radical scavenging rate of 53.35% - 63.05%).

[0202] Example 5: Multifunctional egg yolk protein hydrolysates prepared by the third-step hydrolysis (using compound protease) under different process parameters

[0203] The specific steps are as follows:

[0204] (1) Dissolution: After grinding the supercritical CO2 defatted egg yolk powder, it was sieved through a 60-mesh sieve and mixed with deionized water at a ratio of 1:10 (w / v) and stirred evenly to obtain a defatted egg yolk powder suspension;

[0205] (2) Ultrasonic pretreatment: The defatted egg yolk powder suspension was subjected to ultrasonic pretreatment with a power of 300 W, a reaction frequency of 20 kHz, an ultrasonic mode of 3 s on and 3 s off, and an ultrasonic time of 10 min;

[0206] (3) First step of enzymatic hydrolysis: the pH value of the defatted egg yolk powder suspension was adjusted to 7.5 with 1 mol / L sodium hydroxide solution, pancreatic enzyme was added, the enzyme to substrate (supercritical CO2 defatted egg yolk powder) ratio (E / S) was 1.0%, the enzymatic hydrolysis temperature was 37°C, and the enzymatic hydrolysis time was 2 h;

[0207] (4) Second step of enzymolysis: After the first step of enzymolysis, the pH value of the feed solution was adjusted to 9.9 with a 1 mol / L sodium hydroxide solution, and alkaline protease A was added. The enzyme to substrate (supercritical CO2 defatted egg yolk powder) ratio (E / S) was 2.5%, the enzymolysis temperature was 50° C., and the enzymolysis time was set according to Table 9;

[0208] (5) The third step of enzymatic hydrolysis: When the second step of enzymatic hydrolysis is about to end (without adjusting the pH and temperature), the composite protease is added. The enzymatic hydrolysis process conditions are shown in Table 9.

[0209] (6) Inactivation of enzyme: After the enzymatic hydrolysis is completed, the solution is treated with boiling water for 10 min, cooled to room temperature, and the pH value of the solution is adjusted to 7.0 with 1 mol / L hydrochloric acid;

[0210] (7) Centrifugation: Place the feed liquid in a 50 mL centrifuge tube and centrifuge it at 6000 rpm for 10 min. Concentrate the supernatant in a vacuum and freeze-dry it to obtain the multifunctional egg yolk protein hydrolysate for the determination of biological activity. Collect the lower precipitate, dry it, and use it to determine the yield of soluble enzymatic hydrolysate.

[0211] The yield and biological activity of the multifunctional egg yolk protein hydrolysate from defatted egg yolk powder were determined. The results are shown in Table 9.

[0212] Table 9: Process parameters of compound protease hydrolysis and functional activity of multifunctional egg yolk protease hydrolysate

[0213]

[0214] The results showed that the soluble enzymatic hydrolysate yields of different experimental groups were between 76% and 80%, the ACE inhibition rates were between 68% and 83%, the ferrous ion chelation rates were between 57% and 65%, and the ABTS ·+ The free radical scavenging activity was between 445-466μmol TE / g, and the DPPH free radical scavenging rate was between 51%-55%; the yields and antioxidant activities of different experimental groups were not much different, while the ACE inhibitory activity and ferrous ion chelating activity were quite different.

[0215] When the hydrolysis time of alkaline protease A is 3.5 - 4.5 h, the addition amount of compound protease is 0.6% - 0.9%, and the hydrolysis time of compound protease is 1.5 h, the ACE inhibition rate (80 - 83%), ferrous ion chelation rate (62% - 65%) and antioxidant activity of the hydrolysate are all at the highest level. The yield of the multifunctional egg yolk protein hydrolysate obtained under this hydrolysis process conditions can reach 76.5% - 78.2%, which is higher than the yield of existing egg yolk polypeptides. For example, a hydrolyzed egg yolk powder rich in growth factor-like polypeptides prepared by enzymatic hydrolysis of defatted egg yolk powder as described in the patent with publication number CN119409764A has a yield of 60%, which is much lower than the yield of the hydrolysate of the present invention; the multifunctional egg yolk protein hydrolysate in the present invention simultaneously has high levels of ACE inhibitory activity, metal chelating ability and antioxidant activity, especially the ACE inhibitory activity and antioxidant activity of the prepared multifunctional hydrolysate are much higher than the prior art level. For example, the hydrolysate obtained by Zambrowicz et al. using a non-commercial protease from Asian pumpkin to hydrolyze defatted egg yolk protein has an ACE inhibitory activity IC 50 value of 0.84 mg / mL, while the IC 50 value of the hydrolysate of the present invention has reached 0.45 mg / mL (see Table 14); the ABTS ·+ radical scavenging activity of the hydrolysate obtained by Czelej et al. through two-step enzymatic hydrolysis of ethanol-extracted defatted egg yolk protein with papain and pepsin is 390.43 μmol TE / g, which is significantly lower than the ABTS ·+ radical scavenging activity (445 - 465 μmol TE / g) of the hydrolysate of the present invention (see Tables 9, 14 and 17).

[0216] Using ginger protease, flavor protease, neutral protease and papain for the third-step enzymatic hydrolysis is also applicable when preparing the multifunctional egg yolk protein hydrolysate. After detection, it is found that the levels of various biological activities of the hydrolysate are similar to those of the compound protease used in the third-step enzymatic hydrolysis, so they will not be listed one by one. The range of ginger protease hydrolysis conditions: enzyme to substrate ratio 0.8% - 1.0% (E / S, w / w), hydrolysis pH value 6.8 - 7.2, hydrolysis temperature 63 - 67 °C, hydrolysis time 0.5 - 1 h; the range of flavor protease hydrolysis conditions: enzyme to substrate ratio 0.8% - 1.0% (E / S, w / w), hydrolysis pH value 6.8 - 7.2, hydrolysis temperature 53 - 57 °C, hydrolysis time 0.5 - 1 h; the range of neutral protease hydrolysis conditions: enzyme to substrate ratio 0.8% - 1.0% (E / S, w / w), hydrolysis pH value 6.8 - 7.2, hydrolysis temperature 48 - 52 °C, hydrolysis time 0.5 - 1 h; the range of papain hydrolysis conditions: enzyme to substrate ratio 0.8% - 1.0% (E / S, w / w), hydrolysis pH value 6.8 - 7.2, hydrolysis temperature 48 - 52 °C, hydrolysis time 0.5 - 1 h.

[0217] Example 6: Preparation of multifunctional egg yolk protein hydrolysate using ethanol-extracted defatted egg yolk powder as raw material

[0218] The specific steps are as follows:

[0219] (1) Dissolution: After grinding the supercritical CO2 defatted egg yolk powder, it was sieved through a 60-mesh sieve and mixed with deionized water in a ratio of 1:10 (w / v) and stirred evenly to obtain a defatted egg yolk powder suspension;

[0220] (2) Ultrasonic pretreatment: The defatted egg yolk powder suspension was ultrasonically pretreated with a power of 300 W, a reaction frequency of 20 kHz, an ultrasonic mode of 3 s on and 3 s off, and an ultrasonic time of 10 min;

[0221] (3) First-step enzymatic hydrolysis: The pH value of the defatted egg yolk powder suspension was adjusted to 7.5 with 1 mol / L sodium hydroxide solution, and pancreatin was added. The ratio of enzyme to substrate (ethanol-extracted defatted egg yolk powder) (E / S) was 1.0%, the enzymatic hydrolysis temperature was 37 °C, and the enzymatic hydrolysis time was 2 h;

[0222] (4) Second-step enzymatic hydrolysis: After the first-step enzymatic hydrolysis was completed, the pH value of the material liquid was adjusted to 9.9 with 1 mol / L sodium hydroxide solution, and alkaline protease A was added. The ratio of enzyme to substrate (ethanol-extracted defatted egg yolk powder) (E / S) was 2.5%, the enzymatic hydrolysis temperature was 50 °C, and the enzymatic hydrolysis time was as shown in Table 10;

[0223] (5) Third-step enzymatic hydrolysis: After the second-step enzymatic hydrolysis, compound protease was added (without adjusting the pH value), the enzymatic hydrolysis time was 1.5 h, the enzymatic hydrolysis temperature was 50 °C, and the ratio of enzyme to substrate (ethanol-extracted defatted egg yolk powder) (E / S) was as shown in Table 10;

[0224] (6) Enzyme inactivation: After the enzymatic hydrolysis was completed, it was treated with boiling water for 10 min. After cooling to room temperature, the pH value of the material liquid was adjusted to 7.0 with 1 mol / L hydrochloric acid;

[0225] (7) Centrifugation: The material liquid was placed in a 50 mL centrifuge tube and centrifuged in a centrifuge at a speed of 6000 rpm for 10 min. The supernatant was vacuum concentrated and freeze-dried for the determination of biological activity; the lower-layer precipitate was collected for the determination of the yield of soluble hydrolysate.

[0226] The yield and biological activity of the multifunctional egg yolk protein hydrolysate prepared from ethanol-extracted defatted egg yolk powder were measured, and the results are shown in Table 10.

[0227] Table 10: Yield and biological activity of multifunctional egg yolk protein hydrolysate prepared from ethanol-extracted defatted egg yolk powder

[0228]

[0229] The results showed that the yields of soluble enzymolysates in both experimental groups were above 55%. Since the protein content of defatted egg yolk powder extracted by ethanol was relatively low (about 53%), and the protein content in defatted egg yolk powder by supercritical CO2 could reach 79%, its yield of soluble enzymolysate was lower compared with that of defatted egg yolk powder by supercritical CO2, while the bioactivity levels were similar.

[0230] Since the difference between defatted egg yolk powder extracted by ethanol and that by supercritical CO2 lies in the different degrees of defatting and they are exactly the same in the composition of egg yolk protein, the protease applicable to defatted egg yolk powder extracted by supercritical CO2 and the multifunctional bioactivities of its enzymolysate are also applicable when using defatted egg yolk powder extracted by ethanol as the raw material. After detection, it was found that the bioactivity levels of the enzymolysate were similar to those, which will not be listed one by one.

[0231] Example 7: Screening of macroporous resin types

[0232] 1. Static adsorption and desorption capabilities of different macroporous resin types for bioactive peptides

[0233] (1) Preparation of multifunctional egg yolk protease hydrolysate (Egg Yolk Hydrolysates, EYH):

[0234] After grinding the defatted egg yolk powder by supercritical CO2, it was passed through a 60-mesh sieve and mixed with deionized water in a ratio of 1:10 (w / v) and stirred evenly to obtain a defatted egg yolk powder suspension.

[0235] The defatted egg yolk powder suspension was pretreated by ultrasound with a power of 300 W, a reaction frequency of 20 kHz, an ultrasound mode of 3 s on and 3 s off, and an ultrasound time of 10 min.

[0236] The first enzymatic hydrolysis: The pH value of the defatted egg yolk powder suspension was adjusted to 7.5 with 1 mol / L sodium hydroxide solution, and trypsin was added. The ratio of enzyme to substrate (defatted egg yolk powder by supercritical CO2) (E / S) was 1.0%, the enzymatic hydrolysis temperature was 37 °C, and the enzymatic hydrolysis time was 2 h;

[0237] The second enzymatic hydrolysis: After the first enzymatic hydrolysis was completed, the pH value of the feed liquid was adjusted to 9.9 with 1 mol / L sodium hydroxide solution, and alkaline protease A was added. The ratio of enzyme to substrate (defatted egg yolk powder by supercritical CO2) (E / S) was 2.5%, the enzymatic hydrolysis temperature was 50 °C, and the enzymatic hydrolysis time was 3.5 h;

[0238] The third enzymatic hydrolysis: After the second enzymatic hydrolysis, compound protease was added (without adjusting the pH value). The ratio of enzyme to substrate (defatted egg yolk powder by supercritical CO2) (E / S) was 0.6%, the enzymatic hydrolysis temperature was 50 °C, and the enzymatic hydrolysis time was 1.5 h.

[0239] After the enzymatic hydrolysis was completed, the mixture was treated with boiling water for 10 min. After cooling to room temperature, the material liquid was placed in a 50 mL centrifuge tube (without adjusting the pH value) and centrifuged in a centrifuge at 6000 rpm for 10 min. The supernatant was subjected to vacuum concentration and drying to obtain the multifunctional egg yolk protein hydrolysate.

[0240] (2) Pretreatment of macroporous resin:

[0241] Place different macroporous adsorption resins (physical and chemical properties are shown in Table 3) in absolute ethanol and soak for 4 h to fully swell them. Then wash with distilled water until there is no ethanol smell and keep them in a moist state for standby.

[0242] (3) Static adsorption of macroporous resin:

[0243] Dry the pretreated macroporous adsorption resin in an oven at 60 °C to constant weight. Accurately weigh 5 g each and place them in 250 mL conical flasks. Add 100 mL of absolute ethanol and soak for 12 h to fully expand the resin. Then remove the ethanol, wash with distilled water until there is no ethanol smell, remove the distilled water, and prepare the EYH obtained in step (1) into a 10 mg / mL solution with deionized water. Add 100 mL of the EYH solution to each conical flask and oscillate and adsorb at 150 rpm at room temperature for 3 h. Then collect the supernatant for standby.

[0244] (4) Static desorption of macroporous resin:

[0245] Filter the macroporous resin that has fully adsorbed EYH, wash the resin with an appropriate amount of distilled water to remove the residual solution, place the macroporous resin back into a 250 mL conical flask, add 100 mL of 80% ethanol solution, oscillate and desorb at 150 rpm at room temperature for 3 h, and then collect the supernatant for standby.

[0246] (5) Determination of the adsorption kinetic curve of macroporous resin:

[0247] Different from (3), when oscillating and adsorbing for 0, 4, 8, 12, 16, 20, 25, 30, 40, 50, 60, 80, 100, 120, 150, 180 min, pipette 100 μL of the supernatant for standby.

[0248] 2. Experimental results:

[0249] (1) Determine the peptide content of the supernatant samples adsorbed by the above different resins and calculate the adsorption amount and desorption amount; the results are as Figure 1 shown.

[0250] The results showed that: except for XAD-7HP, the adsorption capacities of the other five macroporous resins were between 95.57 - 123.24 mg / g, the adsorption rates were between 50.30% - 64.86%, the desorption amounts were between 69.48 - 83.62 mg / g, and the desorption rates were between 61.73% - 87.47%.

[0251] (2) Considering the adsorption and desorption capacities of each resin comprehensively, three macroporous resins, DA201-C, XAD-1600, and XAD-16, were selected for the adsorption kinetics study. The peptide contents of the samples adsorbed by the above three resins were determined to further investigate the adsorption properties of the three resins. The results were as Figure 2 shown. The results showed that: the three resins reached the adsorption equilibrium state at about 60 min. The maximum adsorption capacities of DA201-C, XAD-1600, and XAD-16 were 121.50 ± 2.19 mg / g, 117.15 ± 0.68 mg / g, and 100.87 ± 2.63 mg / g respectively. The three macroporous resins were all non-polar resins and all had good adsorption rates (62.76%, 60.51%, and 52.10% respectively) and desorption rates (61.73%, 63.03%, and 87.47% respectively).

[0252] Example 8: Directional separation process of different bioactive peptides

[0253] 1. Separation method of different bioactive peptides in multifunctional egg yolk protein hydrolysate

[0254] (1) Preparation of multifunctional egg yolk protein hydrolysate (EYH):

[0255] Prepared according to the method described in step (1) of Example 7 to obtain EYH.

[0256] (2) Pretreatment of macroporous adsorption resin:

[0257] Immerse the DA201-C macroporous resin in absolute ethanol for 4 h to fully swell it, then wash it with distilled water until there is no ethanol smell and keep it in a moist state for standby.

[0258] (3) Gradient elution:

[0259] The wet method was used for column packing. The pretreated DA201-C resin was loaded into a glass chromatography column (inner diameter d = 2.5 cm × 40 cm). The column was rinsed with deionized water until there was no change detected by the detector at 220 nm. 4 g of EYH obtained in step (1) was weighed and prepared into a 150 mg / mL solution with deionized water, and the loading pH value was adjusted to 8.0. A constant flow pump was used for dynamic loading of the EYH solution. After the loading was completed, the chromatography column was dynamically desorbed with deionized water, 20% ethanol, 40% ethanol, and 80% ethanol in sequence. The loading flow rate and the elution flow rate were both set at 3 mL / min, and the elution volume of each eluent (desorbent) was one column volume (180 mL), as Figure 3 shown. Each elution fraction was collected according to the elution peak order and named water-1, water-2, Ethl-20%, Ethl-40%, and Ethl-80% in sequence. The above eluents were respectively rotary evaporated under vacuum until there was no ethanol left, and then freeze-dried for standby.

[0260] 2. Change the EYH loading amount in step 1(3) to 6 g, and keep the other conditions unchanged.

[0261] (1) The yields and sample recovery rates of each elution fraction were respectively detected, and the results are shown in Table 11, Figure 4 (A).

[0262] Table 11: Yields and sample recovery rates of each component in the gradient elution of macroporous resin under different loading amounts

[0263]

[0264] The results showed that during the dynamic elution process, EYH was separated into five components, namely water-1, water-2, Ethl-20%, Ethl-40%, and Ethl-80%. The sample recovery rates under the two loading amounts were between 85.60% - 88.49%. And with the increase of the loading amount, the yield of water-1 decreased, the yield of water-2 increased, and the yields of other components had little difference.

[0265] (2) The ACE inhibition rate, ferrous ion chelation rate, ABTS ·+ radical scavenging activity, and DPPH radical scavenging rate of each elution fraction were respectively detected, and the results are shown in Figure 4 (B)-(E).

[0266] The results showed that the change of the loading amount had no obvious effect on the biological activities of each elution fraction. Under the two EYH loading amounts, the biological activity trends of each component were the same.

[0267] 3. Change the EYH loading amount and the loading pH value in step 1(3), and keep the other conditions unchanged.

[0268] The sample loading amount of EYH was 6 g, and the sample loading pH values were 6.0, 6.5, 7.0, 7.5, and 8.0 respectively.

[0269] (1) The yields and sample recoveries of each elution fraction were detected respectively, and the results are shown in Table 12 Figure 5 (A).

[0270] Table 12: Yields and sample recoveries of each fraction in gradient elution of macroporous resin at different sample loading pH values

[0271]

[0272] The results showed that there was no significant difference in the sample recoveries among the groups, which were between 87.57% - 88.52%. However, the change in the sample pH value affected the affinity between the peptide and the resin, thus changing the distribution of bioactive peptides among different fractions. As the sample loading pH value increased, the yields of Water-1 and Ethl-20% first decreased and then gradually stabilized, and the yield of Water-2 gradually increased; the yields of Ethl-40% and Ethl-80% showed a weak decreasing and increasing trend respectively, and the sum of the two fractions was about 20%.

[0273] (2) The ACE inhibition rate, ferrous ion chelation rate, ABTS ·+ radical scavenging activity and DPPH radical scavenging rate of each elution fraction were detected respectively; the results are shown in Figure 5 (B)-(E).

[0274] The results showed that within the range of sample loading pH values from 6 to 8, effective separation of different bioactive peptides in EYH could be achieved.

[0275] As Figure 5 (B) shows, the ACE inhibition rates of Ethl-40% and Ethl-80% were significantly improved compared with EYH. Within the range of sample loading pH values from 6 to 8, both Ethl-40% and Ethl-80% could enrich ACE inhibitory peptides. As the sample loading pH value increased, the ACE inhibition rates of the two fractions first increased and then decreased. The ACE inhibition rate of Ethl-80% was the highest at pH 6.5, which increased from 57.53% before separation to 88.84%, and there was no significant difference from the groups with sample loading pH values of 6.0 and 7.5.

[0276] As Figure 5As shown in (C), the ferrous ion chelation rates of Water-1 and Water-2 are greatly improved compared with EYH. In the range of loading pH values from 6 to 8, the elution components of water can enrich metal complex peptides. As the loading pH value increases, the ferrous ion chelation rates of the two components first increase and then decrease. When the pH value is 7.5, the ferrous ion chelation rate of the Water-1 component is the highest, increasing from 59.19% to 76.57%, which is significantly higher than that of other loading pH value groups. The ferrous ion chelation rate of the Water-2 component also has a large increase compared with EYH. When the loading pH values are 6.5 and 7.0, the ferrous ion chelation rate of Water-2 is significantly higher than that of other loading pH value groups, and there is no significant difference among the groups with loading pH values of 6.0, 7.5, and 8.0.

[0277] As shown in Figure 5 (D) and (E), the antioxidant activity of the peptides is relatively sensitive to changes in pH value. Antioxidant peptides with ABTS ·+ radical scavenging activity are enriched in the Ethl-20% component. The ABTS·+ radical scavenging activity of this component increases with the increase of the loading pH value. When the pH value is 8.0, the TEAC value of the Ethl-20% component reaches the highest, increasing by 47% compared with EYH, and there are significant differences among all groups (P < 0.05). Antioxidant peptides with DPPH radical scavenging activity are enriched in the Water-1 component. The DPPH radical scavenging rate of this component increases with the increase of the loading pH value and reaches the highest at pH 8.0, which is significantly higher than that of other groups (P < 0.05), increasing from 25.14% before separation to 52.02%.

[0278] (3) Detect the pH values of each elution component respectively

[0279] As shown in Figure 6 , as the ethanol concentration used for elution increases, the solution pH value of the component gradually increases; as the pH value of the sample before separation increases, the solution pH value of the same component gradually increases.

[0280] Combined with the binding yield and bioactivity measurement results, good separation effects are obtained at loading pH values of 6.5, 7.5, and 8.0. However, considering that the pH values of the ethanol elution components at loading pH values of 7.5 and 8.0 are higher than 7.5, which may affect the taste, the elution components at loading pH values of 6.5, 7.5, and 8.0 are selected for dispersion stability and sensory evaluation.

[0281] (4) Detect the dispersion stability of each elution component respectively

[0282] The results showed that when placed at room temperature for 24 h, the dispersion stability was better when the loading pH values were 7.5 and 8.0. When the loading pH value was 6.5, the dispersion stabilities of Water-1, Water-2, and Ethl-20% were the worst. When placed at room temperature for 48 h, a small amount of precipitation appeared at the bottom of Ethl-40% and Ethl-80% under the three loading pH values. The overall dispersion stability at the loading pH values of 7.5 and 8.0 was better than that at 6.5.

[0283] (5) Detect the sensory evaluation of each elution component respectively

[0284] The results are as Figure 8 shown. The sensory evaluation results of each bioactive peptide component were analyzed separately:

[0285] Water-1: The comprehensive score of the group with a loading pH value of 7.5 was the highest, followed by the group with a pH of 8.0, and the group with a pH of 6.5 had the lowest score. In terms of smell, the group with a pH of 7.5 was better than the group with a pH of 8.0, with less sour / alkaline smell and fishy smell; in terms of state, taste, and overall acceptance, the group with a pH of 8.0 was better than the group with a pH of 7.5.

[0286] Water-2: There was no difference in the comprehensive scores between the groups with loading pH values of 7.5 and 8.0, and the group with a pH of 6.5 had the lowest score. In terms of taste, the score of the group with a pH of 8.0 was slightly higher than that of the group with a pH of 7.5; in terms of smell, the group with a pH of 7.5 was better than the group with a pH of 8.0; there was basically no difference between the two groups in terms of appearance, state, and overall acceptance.

[0287] Ethl-20%: There was no significant difference in the scores of each item of each sample under the three loading pH values.

[0288] Ethl-40%: The comprehensive score of the group with a loading pH value of 6.5 was the highest, followed by the group with a pH of 7.5, and then the group with a pH of 8.0. In terms of smell, the group with a pH of 7.5 was better than the group with a pH of 8.0, with less sour / alkaline smell and fishy smell; for taste, compared with the group with a pH of 8.0, the group with a pH of 7.5 had less fishy smell and bitterness; in terms of overall acceptance, the group with a pH of 7.5 was higher than the groups with a pH of 8.0 and 6.5.

[0289] Ethl-80%: The comprehensive score of the group with a loading pH value of 6.5 was the highest, followed by the group with a pH of 7.5, and then the group with a pH of 8.0. In terms of taste and overall acceptance, the group with a pH of 8.0 was better than the group with a pH of 7.5, and there was basically no difference between the two groups in other aspects.

[0290] Based on the above experimental results, when the loading pH value was between 7.5 and 8.0, the sample recovery rate was between 87% and 89%, with good biological activity and dispersion stability, less peculiar smell, and higher overall acceptance.

[0291] 4. Comparison of the directional separation effects of different bioactive peptides in egg yolk protein hydrolysate before and after omitting the 40% ethanol elution process

[0292] The specific steps are the same as those in Step 1, except that Step (3) is adjusted as follows:

[0293] Use wet method to pack the column. Load the pretreated DA201-C resin into a glass chromatography column (inner diameter d = 2.5 cm × 40 cm), and rinse the column with deionized water until there is no change in the detector at 220 nm. Weigh 6 g of EYH obtained in Step (1), prepare it into a 150 mg / mL solution with deionized water, adjust the loading pH value to 7.5, and perform dynamic loading of the EYH solution with a constant flow pump. After the loading is completed, perform dynamic desorption of the chromatography column with deionized water, 20% ethanol, and 80% ethanol in sequence. The loading flow rate and elution flow rate are both set to 3 mL / min, and the elution volume of each eluent is one column volume (180 mL). Collect each elution fraction in the order of elution peaks, and name them Water-1, Water-2, Ethl-20%, and Ethl-80% in sequence. Vacuum rotary evaporate the above eluents until there is no ethanol, and freeze-dry them for later use.

[0294] Experimental results:

[0295] (1) Measure the yields of Ethl-40% and Ethl-80% before omission and Ethl-80% after omission respectively. The results are as shown in Figure 9 (A).

[0296] According to the bioactivity measurement results in the above embodiments, considering that both Ethl-40% and Ethl-80% are ACE inhibitory peptides, for cost savings, Ethl-80% can be directly used for elution in actual production as the enriched fraction of ACE inhibitory peptides, that is, after eluting one column volume with 20% ethanol, directly elute one column volume with 80% ethanol. As shown in Figure 9 (A), there is no difference between the sum of the yields of Ethl-40% and Ethl-80% before omission and the yield of Ethl-80% after omission, and their yields are 21.24% and 21.39% respectively.

[0297] (2) Measure the ACE inhibitory rates of Ethl-40% and Ethl-80% before omission and Ethl-80% after omission respectively. The results are as shown in Figure 9 (B).

[0298] The results show that, as shown in Figure 9As shown in (B), the ACE inhibition rates of the omitted Ethl-40% and Ethl-80% were 79.08% and 86.40% respectively. After omitting the Ethl-40% elution step and directly using Ethl-80% for elution to obtain the combined fraction, its ACE inhibition rate was 84.10%, indicating that the ACE inhibitory peptide was still eluted in this fraction without causing a significant impact on the activity.

[0299] 5. Method for directional separation of different bioactive peptides in defatted egg yolk powder hydrolysate at different loading concentrations

[0300] The specific steps are the same as those in Step 1, except that Step (3) is adjusted to:

[0301] Use wet method to pack the column. Load the pretreated DA201-C resin into a glass chromatography column (inner diameter d = 2.5 cm × 40 cm), and rinse the column with deionized water until there is no change in the detector at 220 nm. Weigh 6 g of EYH obtained in Step (1), prepare solutions with concentrations of 150, 200, and 250 mg / mL using deionized water, adjust the loading pH value to 7.5, and perform dynamic loading of the EYH solution with a constant flow pump. Dynamically desorb the chromatography column with deionized water, 20% ethanol, and 80% ethanol in sequence. The loading flow rate and elution flow rate are both set to 3 mL / min, and the elution volume of each eluent is one column volume (180 mL). Collect each eluted fraction in the order of elution peaks and name them water-1, water-2, Ethl-20%, and Ethl-80% in sequence. Vacuum rotary evaporate the above eluents until there is no ethanol, and freeze-dry for later use.

[0302] Experimental results:

[0303] (1) Measure the yields and sample recoveries of each eluted fraction, and the results are shown in Table 13, Figure 10 (A).

[0304] Table 13: Yields and sample recoveries of each fraction in macroporous resin gradient elution at different loading concentrations

[0305]

[0306] The results show that there is no significant difference in the sample recoveries among the groups, and the sample recoveries are between 88.22% - 89.37% at the three loading concentrations. As the sample concentration increases, the yield of water-1 gradually decreases, the yield of water-2 gradually increases, and there is no significant difference among the components at 200 mg / mL and 250 mg / mL, indicating that the increase in sample concentration enhances the adsorption capacity of the resin and the unadsorbed water-1 decreases; there is no significant difference in the yields of Ethl-20% and Ethl-80% in each group. Therefore, select the water-1 and water-2 fractions to measure the corresponding bioactivities.

[0307] (2) Measure the ferrous ion chelating rate and DPPH free radical scavenging rate of Water-1 and Water-2 obtained in step (1), and the results are as shown in Figure 10 (B) and (C).

[0308] The results show that the change in the loading concentration has no significant effect on the ferrous ion chelating rate of Water-1. As the concentration of the sample before separation increases, the ferrous ion chelating rate of Water-2 decreases significantly (P < 0.05). The DPPH scavenging rate of Water-1 first decreases and then remains unchanged, and the DPPH scavenging rate of Water-2 shows no significant change.

[0309] Example 9: Comparison of the functional activities of the active peptides obtained in the present invention with the biological activities of the control examples

[0310] I. Preparation of samples

[0311] 1. Preparation method of EYH: Prepared according to the method described in step (1) of Example 7 to obtain EYH.

[0312] 2. Method for directional separation of different bioactive peptides in EYH:

[0313] (1) Pretreatment of macroporous adsorption resin: Immerse the DA201-C macroporous resin in absolute ethanol for 4 h to fully expand it, then wash it with distilled water until there is no ethanol smell and keep it in a moist state for standby.

[0314] (2) Gradient elution: Pack the pretreated DA201-C resin into a glass chromatography column (inner diameter d = 2.5 cm × 40 cm) by wet packing method, wash the column with deionized water until there is no change detected by the detector at 220 nm. Weigh 6 g of EYH, prepare it into a 150 mg / mL solution with deionized water, and adjust the loading pH value to 7.5. Use a constant flow pump to perform dynamic loading of the EYH solution at a loading flow rate of 3 mL / min. After the loading is completed, perform dynamic desorption of the chromatography column with deionized water, 20% ethanol, and 80% ethanol in sequence. The elution flow rate is 4 mL / min, and the elution volume of each eluent is one column volume (180 mL). Collect each elution fraction in the order of elution peaks and name them Water-1, Water-2, Ethl-20%, and Ethl-80% respectively. Vacuum rotary evaporate the above eluents until there is no ethanol, and then freeze-dry them for standby.

[0315] II. Experimental results

[0316] Detect the ACE inhibitory activity IC 50 value of EYH and the Ethl-80% fraction; measure the ferrous ion chelating ability of EYH, Water-1, and Water-2 fractions; measure ABTS of EYH and the Ethl-20% fraction ·+Free radical scavenging activity (TEAC value); determination of the DPPH free radical scavenging activity IC of EYH and water-1 fraction 50 value. Determination of the proliferative activity of EYH, water-1, water-2, Ethl-20%, and Ethl-80% fractions on MC3T3-E1 cells; the results are shown in Table 14 and Figure 11 as follows:

[0317] Table 14: Comparison of the functional activities of egg yolk protease hydrolysates and active peptide fractions, and egg yolk protease hydrolysates or active peptides reported in the literature

[0318]

[0319] 1 Control Example 1: The data is from the published paper YOUSR M, HOWELL N. Antioxidant and ACEInhibitory Bioactive Peptides Purified from Egg Yolk Proteins[J]. International Journal of Molecular Sciences, 2015, 16(12):29161 - 29178.

[0320] 2 Control Example 2: The data is from the published paper ZAMBROWICZ A, ECKERT E, POKORA M, etal. Biological Activity of Egg-Yolk Protein by-Product Hydrolysates Obtainedwith the Use of Non-Commercial Plant Protease[J]. ITALIAN JOURNAL OF FOODSCIENCE, 2015, 27(4):450 - 458.

[0321] 3 Control Example 3: The data is from the published paper CZELEJ M, CZERNECKI T, GARBACZ K, et al. EggYolk as aNewSource of Peptides with Antioxidant and Antimicrobial Properties[J]. Foods, 2023, 12(18):3394.

[0322] 4Control Example 4: The data is from the published paper Song Lushan, Song Li, Zhu Linxian, et al. Preparation and Activity Analysis of Ferrous-Binding Phosvitin Peptide [J]. Journal of Tianjin University of Science & Technology, 2023, 38(05): 1-7.

[0323] 5 Control Example 5: The data is from the published patent Huang Qian, Cui Rui, Liu Wei. An Egg-Source Bifunctional Bioactive Peptide and Its Preparation Method and Application [P]. Hubei Province: CN117843718A, 2024-04-09.

[0324] The results showed that both EYH and each separated component had different biological activities, achieving the simultaneous separation of multiple bioactive peptides. Water-1 was a component enriched with osteogenic active peptides, and it also had antioxidant and metal chelating functions. According to the research results, the main component of Water-1 was phosvitin phosphopeptide, which was derived from phosvitin. Phosvitin was a type of phosphorylated protein in egg yolk and was the protein with the highest known phosphorylation degree in nature. It contained about 50% serine, and more than 90% of the serine was phosphorylated, carrying a large number of negative charges, making it have good bone health activity, antioxidant activity, and the ability to promote calcium absorption (metal chelating ability).

[0325] Ethl-20% was a component enriched with antioxidant peptides. Compared with EYH before separation, the ABTS ·+ radical scavenging activity of Ethl-20% increased by 45%. In Control Example 3, the hydrolysate obtained by Czelej et al. through two-step enzymatic hydrolysis of ethanol-degreased egg yolk protein with papain and pepsin had antioxidant and antibacterial activities, and its ABTS ·+ radical scavenging activity was 390.43 mmol TE / kg (i.e., 390.43 μmol TE / g), which was lower than the ABTS ·+ radical scavenging activities of the enzymatic hydrolysates and separated products in this study.

[0326] Ethl-80% was a component enriched with ACE inhibitory peptides. Compared with EYH before separation, the ACE inhibitory activity of Ethl-80% increased by 73%. In Control Example 1, the ACE inhibitory activity IC 50 value of the EYGF-56 component obtained by Yousr et al. through hydrolysis of defatted egg yolk with pepsin and pancreatin, followed by ultrafiltration and gel filtration chromatography, was 3.35 mg / mL, which was much lower than the ACE inhibitory activities of the enzymatic hydrolysates and separated products of this product; in Control Example 2, the hydrolysate obtained by Zambrowicz et al. through hydrolysis of defatted egg yolk protein with a non-commercial protease from Cucurbita ficifolia had antioxidant activity and ACE inhibitory activity, and its ACE inhibitory activity IC 50The value is 837.75 μg / mL, i.e., 0.84 mg / mL, which is also lower than the enzymolysis products and separated products of this product.

[0327] Combined with the previous experimental results, considering that the activity of water-2 component is relatively low and there is no obvious improvement compared with EYH, water-2 is not used as a functional active component and is only used as an ordinary egg yolk peptide.

[0328] In Comparative Example 4, Song Lushan et al. used a combined enzyme hydrolysis of phosvitin with alkaline protease first and then trypsin. After ultrafiltration, the phosvitin peptide with a relative molecular mass < 3000 had ferrous ion chelating activity and ABTS antioxidant activity; in Comparative Example 5, Huang Qian et al. used egg yolk as the raw material, and after composite enzyme hydrolysis with alkaline protease and trypsin, the P2 component obtained by anion exchange chromatography was a dual-functional bioactive peptide from eggs with calcium absorption promotion and ABTS antioxidant activities; in Comparative Examples 4 and 5, the first two steps of the three-step enzyme hydrolysis method used in this study used the same proteases but with different addition sequences; compared with Comparative Example 4, this study discovered more than 3 kinds of bioactive peptides in defatted egg yolk (increased ACE inhibitory activity, osteoblast proliferation promotion activity, and DPPH scavenging activity); compared with Comparative Example 5, this study increased ACE inhibitory activity and osteoblast proliferation promotion activity. This study greatly improved the utilization rate of defatted egg yolk protein, and the separation technology has strong enrichment ability for bioactive peptides and low cost, which is beneficial to industrial production.

[0329] Example 10: Molecular weight distribution of the multifunctional egg yolk protease hydrolysate and several bioactive peptides in the present invention

[0330] I. Preparation of samples

[0331] The steps are the same as those in Example 9.

[0332] II. Experimental results

[0333] The molecular weight distributions of the multifunctional egg yolk protease hydrolysate EYH and various bioactive peptides in the present invention were detected respectively, and the results are shown in Table 15:

[0334] Table 15: Molecular weight distributions of the multifunctional egg yolk protease hydrolysate and various bioactive peptides

[0335]

[0336] The results showed that the egg yolk peptides with a molecular weight less than 1 kDa in EYH accounted for more than 81%; the egg yolk peptides with a molecular weight less than 1 kDa in the ordinary egg yolk peptide (water-2) accounted for 84.8%; the molecular weight distributions of the three bioactive peptides were as follows:

[0337] In the osteogenic active peptide, the egg yolk peptides with a molecular weight less than 1 kDa accounted for more than 80%, and among them, the molecular weight less than 500 Da accounted for 56.8%;

[0338] Among the antioxidant peptides, the egg yolk protein peptides with a molecular weight less than 1 kDa account for more than 80%, and those with a molecular weight less than 500 Da account for 55.4%;

[0339] Among the ACE inhibitory peptides, the egg yolk protein peptides with a molecular weight less than 1 kDa account for 76%, and those with a molecular weight less than 500 Da account for 48%.

[0340] Example 11: Amino acid composition of the multifunctional egg yolk protein hydrolysate and various bioactive peptides in the present invention

[0341] I. Preparation of samples

[0342] The steps are the same as those in Example 9.

[0343] II. Experimental results

[0344] The amino acid compositions of the multifunctional egg yolk protein hydrolysate and various bioactive peptides were detected respectively, and the results are shown in Table 16:

[0345] Table 16: Amino acid composition of the multifunctional egg yolk protein hydrolysate and various bioactive peptides (relative content, %)

[0346]

[0347]

[0348] n.d.: Not detected

[0349] a HAAs: Hydrophobic amino acids, including Ala, Pro, Val, Met, Ile, Leu and Phe.

[0350] b NCAAs: Negatively charged amino acids, including Asp and Glu

[0351] c PCAAs: Positively charged amino acids, including His, Arg and Lys.

[0352] The results showed that EYH contained relatively high levels of Ile and Lys, with relative contents reaching 17.70% and 15.35% respectively. As the ethanol concentration increased during elution, the hydrophobic amino acids in the components gradually increased while the hydrophilic amino acids gradually decreased. The proportion of hydrophilic amino acids in the osteogenic active peptide (Water-1) could reach 88.72%, and the hydrophobic amino acids in the ACE inhibitory peptide (Ethl-80%) accounted for approximately 50%, conforming to the structural characteristics of ACE inhibitory peptides. The serine content in Water-1 was as high as 22.23%, indicating that the main component of this fraction was phosvitin phosphopeptide. The antioxidant peptide (Ethl-20%) was rich in typical amino acids with free radical scavenging activity such as Cys, Lys, Tyr, Ser, and His, conforming to the amino acid characteristics of antioxidant peptides.

[0353] Example 12: Simultaneous separation and enrichment of multiple bioactive peptides from ethanol-extracted defatted egg yolk powder

[0354] The specific implementation method was the same as that of Example 9, except that the raw material was adjusted to: ethanol-extracted defatted egg yolk powder;

[0355] Multiple egg yolk bioactive peptides were separately prepared: Water-1 (multifunctional bone health peptide), Water-2 (ordinary egg yolk peptide), Ethl-20% (antioxidant peptide), and Ethl-80% (ACE inhibitory peptide).

[0356] The results are shown in the following table:

[0357] Table 17: Effects of simultaneous separation and enrichment of multiple bioactive peptides from ethanol-extracted defatted egg yolk powder

[0358]

[0359] The results showed that there was no significant difference between the multifunctional hydrolysate prepared from ethanol-extracted defatted egg yolk powder and that prepared from supercritical CO2 egg yolk powder, except that the yield of egg yolk protein peptides was slightly lower. By using macroporous resin chromatography columns for adsorption and gradient elution of bioactive peptides, the test results showed that the enrichment and separation effects were still very good, and the activity values of the three active peptides obtained were not significantly different from those of the bioactive peptides prepared from supercritical CO2 egg yolk powder. The enzymatic hydrolysis method and the method of enriching and separating bioactive peptides using macroporous resins used in the previous examples were also applicable to ethanol-extracted defatted egg yolk powder.

[0360] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A preparation method of a multifunctional egg yolk protease hydrolysate, characterized in that, The method comprises the following steps: (1) Dissolution: After pulverizing defatted egg yolk powder, it is mixed with water at a mass-to-volume ratio of 1:10 to 1:7 to obtain a defatted egg yolk powder suspension; (2) Ultrasonic pretreatment: The defatted egg yolk powder suspension obtained in step (1) is subjected to ultrasonic pretreatment; (3) First enzymatic hydrolysis: Trypsin is added to the pretreated defatted egg yolk powder suspension obtained in step (2) for enzymatic hydrolysis; (4) Second enzymatic hydrolysis: After the first enzymatic hydrolysis ends, alkaline protease A is added for enzymatic hydrolysis; (5) Third enzymatic hydrolysis: After the second enzymatic hydrolysis ends, protease is added for enzymatic hydrolysis, and the protease includes: compound protease, ginger protease, flavor protease, neutral protease, papain; (6) Preparation of multifunctional egg yolk protein hydrolysate: After the enzymatic hydrolysis ends, the enzyme is inactivated, and the supernatant is taken by centrifugation or filtration. The supernatant is concentrated and dried to obtain the product.

2. The preparation method of the multifunctional egg yolk protein hydrolysate according to claim 1, wherein In step (1), the defatted egg yolk powder is supercritical CO2 defatted egg yolk powder or ethanol-extracted defatted egg yolk powder; the conditions of ultrasonic treatment are: power is 250 - 350 W, reaction frequency is 15 - 25 kHz, and time is 5 - 20 min; Preferably, the enzyme for the first enzymatic hydrolysis is trypsin, and the enzyme for the second enzymatic hydrolysis is alkaline protease A; the enzymatic hydrolysis conditions for adding trypsin are: enzyme-to-substrate ratio 1% - 2% (E / S, w / w), enzymatic hydrolysis pH value 7.0 - 7.5, enzymatic hydrolysis temperature 37 °C, enzymatic hydrolysis time 2 - 4 h; the enzymatic hydrolysis conditions for adding alkaline protease A are: enzyme-to-substrate ratio 2.0% - 2.5% (E / S, w / w), enzymatic hydrolysis pH value 9.5 - 10.0, enzymatic hydrolysis temperature 48 - 52 °C, enzymatic hydrolysis time 3 - 5 h; Preferably, the enzyme for the third enzymatic hydrolysis is compound protease, and the enzymatic hydrolysis conditions are: enzyme-to-substrate ratio 0.6% - 0.9% (E / S, w / w), enzymatic hydrolysis pH value 6.8 - 7.2, enzymatic hydrolysis temperature 48 - 52 °C, enzymatic hydrolysis time 1 - 2 h; Preferably, the enzyme for the third enzymatic hydrolysis is ginger protease, and the enzymatic hydrolysis conditions are: enzyme-to-substrate ratio 0.8% - 1.0% (E / S, w / w), enzymatic hydrolysis pH value 6.8 - 7.2, enzymatic hydrolysis temperature 63 - 67 °C, enzymatic hydrolysis time 0.5 - 1 h; Preferably, the enzyme for the third enzymatic hydrolysis is flavor protease, and the enzymatic hydrolysis conditions are: enzyme-to-substrate ratio 0.8% - 1.0% (E / S, w / w), enzymatic hydrolysis pH value 6.8 - 7.2, enzymatic hydrolysis temperature 53 - 57 °C, enzymatic hydrolysis time 0.5 - 1 h; Preferably, the enzyme for the third enzymatic hydrolysis is neutral protease, and the enzymatic hydrolysis conditions are: enzyme-to-substrate ratio 0.8% - 1.0% (E / S, w / w), enzymatic hydrolysis pH value 6.8 - 7.2, enzymatic hydrolysis temperature 48 - 52 °C, enzymatic hydrolysis time 0.5 - 1 h; Preferably, the enzyme for the third enzymatic hydrolysis is papain, and the enzymatic hydrolysis conditions are: enzyme-to-substrate ratio 0.8% - 1.0% (E / S, w / w), enzymatic hydrolysis pH value 6.8 - 7.2, enzymatic hydrolysis temperature 48 - 52 °C, enzymatic hydrolysis time 0.5 - 1 h.

3. The multifunctional egg yolk protein hydrolysate prepared by the preparation method according to claim 1 or 2.

4. A method for simultaneously enriching and separating multiple bioactive peptides, characterized in that, The method comprises the following steps: (1) The multifunctional egg yolk protein hydrolysate prepared by the method according to claim 1 or 2, or the multifunctional egg yolk protein hydrolysate according to claim 3, is redissolved in water to obtain a sample; the obtained sample is loaded and adsorbed onto a macroporous resin chromatography column; the macroporous resin includes, but is not limited to, DA201-C, XAD-1600, and XAD-16 macroporous resins; (2) Gradient elution: The chromatography column is dynamically desorbed successively with deionized water, 20% aqueous ethanol solution, 40% aqueous ethanol solution, and 80% aqueous ethanol solution, and each elution fraction is collected in the order of elution peaks and named water-1 fraction, water-2 fraction, 20% ethanol fraction, 40% ethanol fraction, and 80% ethanol fraction in turn; Or, the chromatography column is dynamically desorbed successively with deionized water, 20% aqueous ethanol solution, and 80% aqueous ethanol solution, and each elution fraction is collected in the order of elution peaks and named water-1 fraction, water-2 fraction, 20% ethanol fraction, and 80% ethanol fraction in turn; (3) Each elution fraction is concentrated and dried respectively to obtain each bioactive peptide fraction; the water-1 obtained after drying is the osteogenic active peptide, the water-2 obtained after drying is the ordinary egg yolk peptide, the 20% ethanol fraction obtained after drying is the antioxidant peptide, and the 40% and 80% ethanol fractions obtained after drying are the ACE inhibitory peptides.

5. The method for simultaneously enriching and separating multiple bioactive peptides according to claim 4, wherein, In step (1), the sample concentration is a solution of 150 - 250 mg / mL, and the loading pH value is 6.5 - 8.

0.

6. A plurality of bioactive peptides prepared by the method according to claim 4 or 5, characterized in that, The multiple bioactive peptides are osteogenic active peptides, ordinary egg yolk peptides, antioxidant peptides, and ACE inhibitory peptides.

7. Use of the osteogenic active peptide according to claim 6 as a functional ingredient in the preparation of foods, functional foods, and / or health foods having the function of promoting bone growth or improving bone density.

8. Use of the antioxidant peptide according to claim 6 as a functional ingredient in the preparation of antioxidant foods, functional foods, and / or health foods.

9. Use of the ACE inhibitory peptide according to claim 6 as a functional ingredient in the preparation of health foods or functional foods for assisting in reducing hypertension.

10. The application according to any one of claims 7 to 9, characterized in that, The health food also contains excipients; the dosage form of the health food can be solid powder, beverage, capsule, or tablet.

Images