Preparation method and application of egg yolk polypeptide with ACE (Angiotensin Converting Enzyme) inhibitory activity

Abstract

The invention relates to a preparation method and application of yolk polypeptide with ACE inhibitory activity, and the method comprises the following steps: Q1, adding degreased yolk powder into a proper amount of water, dissolving, and adjusting the pH value to 6-8; and Q2, adding alkaline protease or flavourzyme to carry out enzymolysis for 1.5-2.5 hours under the conditions that the temperature is 50-60 DEG C and the pH value is 7-8, and then carrying out enzyme deactivation to obtain the egg yolk hydrolysate. The ACE inhibitor with high activity is prepared by adopting step-by-step enzyme hydrolysis, five ACE inhibitory peptides (KFIPL, KFLPGY, KFLPTF, APTFGKL and KLPDMILY) with high activity are screened and identified by utilizing a bioinformatics method, and in order to solve the problem that the polypeptide is unstable after being sterilized in the functional beverage, CMC and GG are used as stabilizers, so that the utilization problem of the enzymolysis polypeptide in the functional beverage is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a preparation method and application of an egg yolk polypeptide with ACE inhibitory activity. Background Art

[0002] Hypertension (Hypertension) poses an increasingly serious threat to human health and is a major risk factor for myocardial infarction, heart failure, renal failure, stroke, and even death. Angiotensin-converting enzyme (ACE) is a key enzyme in blood pressure regulation, catalyzing the conversion of angiotensin I to the highly active angiotensin II, one of the strongest vasoconstrictors. This vasoconstriction reduces vascular lumen diameter, increases peripheral resistance, and thus elevates blood pressure. Therefore, inhibiting ACE activity is considered an effective strategy for lowering blood pressure. Although synthetic ACE inhibitors such as captopril, lisinopril, and enalapril are widely used clinically, long-term use is often associated with adverse reactions such as cough, headache, and taste disturbances. Therefore, the development of safe, non-toxic, and widely available natural ACE inhibitors has become a hot topic in recent research in the field of functional foods and medicinal foods.

[0003] Eggs contain all the nutrients necessary for embryonic development, with up to 98% of their protein being bioavailable to the human body. Egg yolk, a nutritious food with a high protein content, has attracted increasing attention in recent years in the research of bioactive peptides. Egg yolk protein is primarily composed of yolk globulin (38% in α, β, and γ forms), yolk lipophosphoprotein (36% in α and β forms), low-density lipoprotein (LDL) (17%), and poly(vinylpyrrolidone) (PV) (8-9%). Existing research indicates that egg yolk protein, particularly poly(vinylpyrrolidone) and low-density lipoprotein (LDL), has high bioavailability and a unique amino acid profile, showing great potential for the extraction of bioactive peptides. Therefore, developing safe and effective food-derived antihypertensive peptides, and subsequently functional foods, using egg yolk as a raw material is crucial for increasing the utilization of egg yolk resources and balancing the development of the egg industry.

[0004] However, the traditional enzymatic hydrolysis method for preparing peptides has problems such as single enzyme selection and low efficiency of functional peptide release, making it difficult to obtain highly active target peptides. At the same time, the obtained peptides have poor stability during processing, especially under treatment conditions such as high temperature and pasteurization, which are prone to precipitation, turbidity and reduced functional activity, limiting their promotion and application in liquid foods such as beverages. In addition, there is currently a lack of systematic screening and verification of the relationship between the structure and activity of functional peptides, resulting in unstable functional effects of the developed products and affecting the industrialization process. Therefore, there is an urgent need to develop an efficient peptide preparation and purification strategy, and at the same time combine it with food formula design to improve its stability and practicality in liquid products. Summary of the Invention

[0005] In view of the above problems, the present invention provides a preparation method and application of egg yolk polypeptides with ACE inhibitory activity. It not only solves the problem of polypeptide instability after sterilization in functional beverages, but also uses CMC and GG as stabilizers to solve the problem of enzymatic polypeptide utilization in functional beverages. In addition, it adopts step-by-step enzymatic hydrolysis to prepare highly active ACE inhibitors, and uses bioinformatics methods with the help of an online active peptide database to screen out 5 new ACE inhibitory peptides from enzymatically hydrolyzed egg yolks, and elucidate the inhibition mechanism.

[0006] In order to achieve the above-mentioned and other related objectives, the present invention provides the following technical solution: a method for preparing an egg yolk polypeptide having ACE inhibitory activity, the method comprising: Q1. Dissolve defatted egg yolk powder in water and adjust the pH to 6-8. Q2. Add alkaline protease or flavor protease and perform enzymatic hydrolysis at 50-60°C and pH 7-8 for 1.5-2.5 hours, then inactivate the enzyme to obtain egg yolk hydrolyzate. Q3. Add flavor protease or alkaline protease to the egg yolk hydrolyzate, continue enzymatic hydrolysis at 50-60°C, pH = 7-8 for 1.5-2.5h, and then inactivate the enzyme to obtain an egg yolk enzymatic hydrolyzate; Q4. freeze-drying the egg yolk enzymatic hydrolysate to obtain egg yolk polypeptide.

[0007] Preferably, the amount of alkaline protease used is 2000-4000 U / g.

[0008] Preferably, the amount of flavor protease used is 2000-4000 U / g.

[0009] Preferably, the egg yolk enzymatic hydrolysate is passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain a first filtrate with a molecular weight of less than 1 kDa, which is prepared into a freeze-dried powder for later use.

[0010] The egg yolk polypeptide prepared by the above method has an amino acid sequence of any one or more of the following combinations: KFIPL, KFLPGY, KFLPTF, APTFGKL, KLPDMILY.

[0011] A peptide segment, wherein the peptide segment is used to prepare an ACE-inhibiting drug or a food having auxiliary ACE-inhibiting function, or to measure the ACE-inhibiting effect for purposes other than disease diagnosis and treatment, wherein the amino acid sequence of the peptide segment is any one or more of the following: KFIPL, KFLPGY, KFLPTF, APTFGKL, KLPDMILY.

[0012] The egg yolk polypeptide prepared by the method has ACE inhibition and renin inhibition activities and is used in functional active beverages.

[0013] A functional beverage with ACE inhibitory activity is prepared by adjusting the pH of an egg yolk hydrolysate to 3.5-5.0 using a citric acid / sodium citrate buffer system, pasteurizing it at 80°C for 10 minutes, and rapidly cooling it. The beverage also includes two polysaccharides, orange juice powder, carboxymethyl cellulose (CMC), and guar gum (GG), as stabilizers, at a concentration of 3% by weight of the egg yolk hydrolysate. The CMC constitutes 0.45% of the egg yolk hydrolysate, and the guar gum constitutes 0.30% of the egg yolk hydrolysate.

[0014] The present invention has the following positive effects: 1. The present invention significantly improves the release efficiency of ACE inhibitory active substances by optimizing protease and adopting dual-enzyme synergistic hydrolysis technology. The prepared enzymatic hydrolysate has a high content of small molecule peptides and free amino acids, stable physicochemical properties, and exhibits good solubility, charge uniformity, and structural stability, which is beneficial for maintaining processing performance and biological activity in subsequent applications.

[0015] 2. Through ultrafiltration purification and peptide screening, the present invention successfully screened several small-molecule peptides (KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY) with high ACE inhibitory activity and precisely mapped their mechanisms of action. These functional peptides exhibit high biological activity, low toxicity, and favorable pharmacokinetic properties, demonstrating their potential as functional factors in the development of antihypertensive foods and related formulations.

[0016] 3. The present invention effectively improves the stability of functional polypeptide beverages during the pasteurization process through a polysaccharide compound system. The selected polysaccharides and polypeptides have good charge complementarity and interaction ability, which can enhance the stability of the system structure at the molecular level, extend the shelf life of the product, and improve the overall quality and functional retention ability of the beverage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of (A) DH inhibitory activity of the single-enzyme hydrolysate of the present invention; Figure 2 (B) is a schematic diagram of the ACE inhibitory activity of the single-enzyme hydrolysate of the present invention; Figure 3 Schematic diagram of (A) DH inhibitory activity of the dual-enzyme hydrolysate of the present invention; Figure 4 (B) is a schematic diagram of the ACE inhibitory activity of the dual-enzyme hydrolysate of the present invention; Figure 5 Schematic diagram of the solubility of different enzymatic hydrolysate powders of the present invention; Figure 6Schematic diagram of protein solubility of the present invention; Figure 7 Schematic diagram of the ACE inhibitory activity of the ultrafiltration fraction of the present invention; Figure 8 Schematic diagram of sequencing result analysis of the polypeptide (A) of the present invention; Figure 9 Schematic diagram of analysis of the sequencing results of polypeptide length distribution (B) of the present invention; Figure 10 The molecular docking diagram of the five active ACE inhibitory peptides of the present invention; Figure 11 ACE inhibitory activity of the synthetic peptide; Figure 12 Zeta potential and macroscopic images of the polysaccharide-stabilized beverage of the present invention at different pH values, (A) Zeta potential, (B) macroscopic image after storage at 25°C for 5 days; Figure 13 The particle size distribution and macroscopic images of different polysaccharide-stabilized beverages of the present invention are shown in Figure 1. (A) Particle size distribution, (B) Macroscopic image when stored at 25°C. Figure 14 Schematic diagram of ΔBS values ​​of polysaccharide-stabilized beverages with different ratios of the present invention; Figure 15 (A) Schematic diagram of macro viscosity index of microrheological analysis of polysaccharide-stabilized beverages with different ratios of the present invention; Figure 16 (B) is a schematic diagram of elasticity index of microrheological analysis of polysaccharide-stabilized beverages with different ratios of the present invention; Figure 17 (C) Schematic diagram of the fluidity index of microrheological analysis of polysaccharide-stabilized beverages with different ratios of the present invention. DETAILED DESCRIPTION

[0018] The following description of exemplary embodiments of the present disclosure is made in conjunction with the accompanying drawings, including various details of the embodiments of the present disclosure to facilitate understanding. These details should be considered as merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present disclosure. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0019] Example 1: Preparation of egg yolk polypeptides and screening of target peptides.

[0020] (1) Preparation of defatted egg yolk powder: Egg yolk powder and ethyl acetate were diluted in a ratio of 1:4 (egg yolk powder g: ethyl acetate mL). The ultrasonic temperature was controlled at 0 °C, the ultrasonic power was 100 W, and the ultrasonic treatment time was 15 min (ultrasonication for 3 s, and rest for 3 s). All the ultrasonically treated samples were filtered through a G3 Buchner funnel, and the filter cake was placed in a vacuum drying oven at 40 °C to evaporate the solvent for about 24 h. After that, the defatted egg yolk powder was crushed to obtain the powder for the next step.

[0021] (2) Enzymatic hydrolysis: the egg yolk powder obtained in step (1) is dispersed in deionized water to prepare a suspension with a mass fraction of 5% (egg yolk powder / water, w / w). 4000U / g alkaline protease is used for enzymatic hydrolysis at pH = 8 and 60°C for 2.5 hours, and the enzyme is inactivated to obtain the egg yolk hydrolyzate. 2000U / g flavor protease is then used for enzymatic hydrolysis at pH = 8 and 55°C for 1.5 hours. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is placed in a boiling water bath for 5 minutes to inactivate the enzyme, and then cooled to obtain an egg yolk enzymatic hydrolyzate for use. The egg yolk enzymatic hydrolyzate is passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain a first filtrate with a molecular weight of <1 kDa, which is prepared into a freeze-dried powder for use.

[0022] (3) Screening and identification of target peptides The egg yolk peptides obtained in step (2) were fractionated by ultrafiltration, and a total of 4 peptide fragments were fractionated. Among them, the peptide <1kDa had the highest activity. Further identification by mass spectrometry identified 559 egg-derived peptides. Source analysis showed that most of the active peptides were derived from PV protein in egg yolk, accounting for 39.4%. Peptide Ranker was used to predict the potential activity of the peptides. 31 peptides were screened out by sequence alignment through Biopep database analysis. The hydrophobicity and toxicity of the peptides were analyzed using ToxinPred, and the results showed that all 31 peptides were non-toxic. 13 peptides with high hydrophobicity were screened for ADMET property analysis and activity prediction. The results showed that KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY had ACE inhibitory and renin inhibitory activities, and had good ADMET properties. Molecular docking was performed on these 5 peptides. Results showed that KFIPL and KFLPGY likely interact with ACE through mixed competitive inhibition, while KFLPTF, APTFGKL, and KLPDMILY interact with ACE through noncompetitive inhibition. All five peptides interacted with Zn 701 through metal coordination or ionic bonding. KLPDMILY formed eight hydrogen bonds with ACE, with the lowest binding energy of -13.539 kal / mol.

[0023] Example 2: Preparation of egg yolk polypeptides and screening of target peptides.

[0024] (1) Preparation of defatted egg yolk powder: Egg yolk powder and ethyl acetate were diluted in a ratio of 1:6 (egg yolk powder g: ethyl acetate mL). The ultrasonic temperature was controlled at 3°C, the ultrasonic power was 200 W, and the ultrasonic treatment time was 25 min (ultrasonication for 3 s, and rest for 3 s). All the ultrasonically treated samples were filtered through a G3 Buchner funnel, and the filter cake was placed in a vacuum drying oven at 40°C to evaporate the solvent for about 24 h. After that, the defatted egg yolk powder was crushed to obtain the powder for the next step.

[0025] (2) Enzymatic hydrolysis: the egg yolk powder obtained in step (1) is dispersed in deionized water to prepare a suspension with a mass fraction of 6% (egg yolk powder / water, w / w). 3000U / g alkaline protease is used for enzymatic hydrolysis at pH = 7 and 50°C for 2h to obtain the egg yolk hydrolyzate. After the enzymatic hydrolysis is completed, the enzyme is inactivated, and then 3000U / g flavor protease is used for enzymatic hydrolysis at pH = 7 and 55°C for 2h. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is placed in a boiling water bath for inactivation of the enzyme for 5min and then cooled to obtain an egg yolk enzymatic hydrolyzate for use. The egg yolk enzymatic hydrolyzate is passed through an ultrafiltration membrane with a pore size of 1kDa to obtain a first filtrate with a molecular weight of <1kDa, which is prepared into a freeze-dried powder for use.

[0026] (3) Screening and identification of target peptides.

[0027] The egg yolk peptides obtained in step (2) were fractionated by ultrafiltration, and a total of 4 peptide fragments were fractionated. Among them, the peptide <1kDa had the highest activity. Further identification by mass spectrometry identified 559 egg-derived peptides. Source analysis showed that most of the active peptides were derived from PV protein in egg yolk, accounting for 39.4%. Peptide Ranker was used to predict the potential activity of the peptides. 31 peptides were screened out by sequence alignment through Biopep database analysis. The hydrophobicity and toxicity of the peptides were analyzed using ToxinPred, and the results showed that all 31 peptides were non-toxic. 13 peptides with high hydrophobicity were screened for ADMET property analysis and activity prediction. The results showed that KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY had ACE inhibitory and renin inhibitory activities, and had good ADMET properties. Molecular docking was performed on these 5 peptides. Results showed that KFIPL and KFLPGY likely interact with ACE through mixed competitive inhibition, while KFLPTF, APTFGKL, and KLPDMILY interact with ACE through noncompetitive inhibition. All five peptides interacted with Zn 701 through metal coordination or ionic bonding. KLPDMILY, with eight peptides, had the lowest binding energy of -13.539 kal / mol.

[0028] Example 3: Preparation of egg yolk polypeptides and screening of target peptides.

[0029] The difference from Example 2 is that: (2) Enzymatic hydrolysis: the egg yolk powder obtained in step (1) is dispersed in deionized water to prepare a suspension with a mass fraction of 6% (egg yolk powder / water, w / w). 2000U / g alkaline protease is used for enzymatic hydrolysis at pH = 7 and 53°C for 2 hours, and the enzyme is inactivated to obtain the egg yolk hydrolyzate. 4000U / g flavor protease is then used for enzymatic hydrolysis at pH = 7.5 and 57°C for 2 hours. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is placed in a boiling water bath for 5 minutes to inactivate the enzyme, and then cooled to obtain an egg yolk enzymatic hydrolyzate for use. The egg yolk enzymatic hydrolyzate is passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain a first filtrate with a molecular weight of <1 kDa, which is prepared into a freeze-dried powder for use.

[0030] Example 4: Preparation of egg yolk polypeptides and screening of target peptides.

[0031] (1) Preparation of defatted egg yolk powder.

[0032] Dilute egg yolk powder and ethyl acetate in a ratio of 1:8 (g egg yolk powder: mL ethyl acetate). Ultrasonication temperature was controlled at 4°C, power at 400 W, and duration for 10 minutes (3 seconds on, 3 seconds off). All ultrasonically treated samples were filtered through a G3 Büchner funnel. The filter cake was placed in a vacuum drying oven at 40°C to evaporate the solvent for approximately 24 hours. The resulting defatted egg yolk powder was then ground and used in the next step.

[0033] (2) Enzymatic hydrolysis of defatted egg yolk powder.

[0034] The egg yolk hydrolyzate was obtained by enzymatic hydrolysis using 2000 U / g flavor protease at pH 8 and 55°C for 2 hours, followed by enzyme inactivation. The egg yolk hydrolyzate was then obtained by enzymatic hydrolysis using 4000 U / g alkaline protease at pH 7.5 and 52°C for 1.5 hours. After completion of the enzymatic hydrolysis, the hydrolyzate was inactivated in a 100°C water bath for 10 minutes, thereby obtaining the egg yolk hydrolyzate. The egg yolk hydrolyzate was then passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain a first filtrate having a molecular weight of <1 kDa, which was then freeze-dried into a powder for later use.

[0035] (3) Screening, identification and synthesis of target peptides Ultrafiltration products with a molecular weight <1 kDa were subjected to HPLC-MS / MS. The mobile phases for liquid chromatography consisted of mobile phase A and mobile phase B. The elution column was a domestic fused silica capillary column (ID 75 µm, 150 mm, Upchurch, Oak Harbor, WA) containing C-18 resin (300Å, 5 µm, Varian, Lexington, MA). Mobile phase A consisted of 0.1% formic acid. Mobile phase B consisted of 0.1% trifluoroacetic acid. The elution program was: 3% B from 0 to 8 min, 3 to 50% B from 8 to 40 min, 50% B from 40 to 55 min, 50% B from 55 to 75 min, and 99% B from 75 to 85 min. The flow rate was 0.3 µL / min, and the injection volume was 1 µL.

[0036] Through mass spectrometry, 559 egg-derived peptides were identified. Source analysis showed that most of the active peptides were derived from PV protein in egg yolk, accounting for 39.4%, and 2-12 amino acids accounted for about 93% (such as Figure 8 or Figure 9 PeptideRanker was used to predict the potential activity of the peptides. Sequence alignment was performed through Biopep database analysis to screen 31 peptides. Hydrophobicity and toxicity analysis using ToxinPred revealed that all 31 peptides were non-toxic.

[0037] Thirteen highly hydrophobic peptides were screened for ADMET property analysis and activity prediction. The results showed that KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY had ACE inhibitory and renin inhibitory activities, and had good ADMET properties.

[0038] The synthetic peptides KFIPL, KFLPGY, KFLPTF, APTFGKL and KLPDMILY were obtained by solid phase synthesis and prepared into lyophilized powder for use.

[0039] Example 5: Preparation of egg yolk polypeptides and screening of target peptides.

[0040] The difference from Example 3 is that: (2) Enzymatic hydrolysis of defatted egg yolk powder.

[0041] The egg yolk hydrolyzate was obtained by enzymatic hydrolysis using 3000 U / g of flavor protease at pH 7 and 50°C for 2 hours, followed by enzyme inactivation. The egg yolk hydrolyzate was then obtained by enzymatic hydrolysis using 3000 U / g of alkaline protease at pH 7.5 and 55°C for 2 hours. After completion of the enzymatic hydrolysis, the hydrolyzate was placed in a 100°C water bath for 10 minutes to inactivate the enzyme, thereby obtaining the egg yolk hydrolyzate. The egg yolk hydrolyzate was then passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain a first filtrate having a molecular weight of <1 kDa, which was then freeze-dried into a powder for later use.

[0042] Example 6: Preparation of egg yolk polypeptides and screening of target peptides.

[0043] The difference from Example 3 is that: (2) Enzymatic hydrolysis of defatted egg yolk powder.

[0044] The egg yolk hydrolyzate was obtained by enzymatic hydrolysis using 4000 U / g flavor protease at pH 7.5 and 50°C for 2 hours, followed by enzyme inactivation. The egg yolk hydrolyzate was then obtained by enzymatic hydrolysis using 2000 U / g alkaline protease at pH 7.5 and 60°C for 2 hours. After completion of the enzymatic hydrolysis, the hydrolyzate was placed in a 100°C water bath for 10 minutes to inactivate the enzyme, thereby obtaining the egg yolk hydrolyzate. The egg yolk hydrolyzate was then passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain a first filtrate having a molecular weight of <1 kDa, which was then freeze-dried into a powder for later use.

[0045] Example 7: Preparation of a beverage with ACE inhibition function The present invention further utilizes the above enzymatic hydrolysate containing highly active peptide segments to prepare a functional beverage.

[0046] Add 3 parts (by weight, 3% of the egg yolk hydrolysate, the same below) of orange powder to 100 parts of egg yolk hydrolysate and stir for 30 minutes. Continue adding different concentrations of CMC and GG to the mixture.

[0047] CMC was added at concentrations of 0.3% (0.3 parts, 0.3% of egg yolk enzymatic hydrolysate, the same below), 0.45% and 0.6%, respectively, and the samples were named "CMC-0.3", "CMC-0.45" and "CMC-0.6".

[0048] GG was added at concentrations of 0.2%, 0.3%, and 0.4%, respectively, and the samples were named “GG-0.2,” “GG-0.3,” and “GG-0.4.”

[0049] A mixture of 0.3% CMC and 0.2% GG was added and the sample was named "CMC+GG". A mixture without polysaccharide was used as a control and was named "control".

[0050] All samples were stirred for 30 minutes. The pH was adjusted with citric acid and sodium citrate. The mixture was pasteurized by storing it at 80°C for 10 minutes and then cooled in an ice bath for 5 minutes.

[0051] In the optimized formula, 3% orange juice powder was added by weight to the enzymatic peptide solution to improve taste. Two polysaccharides, carboxymethyl cellulose (CMC) and guar gum (GG), were then added as stabilizers. Comparative testing determined the optimal blend ratio; for example, 0.45% CMC and 0.30% GG achieved optimal stability. After stirring the mixed solution, the pH was adjusted to 3.5-5.0 using a citric acid-sodium citrate buffer. The solution was then pasteurized by heating in a water bath at 80°C for 10 minutes and rapidly cooled to produce a stable beverage sample.

[0052] Test example: The test method of the data in the embodiment of the present invention is as follows: Determination of hydrolysis degree: In a dark place, dissolve 80 mg of OPA in 2 mL of anhydrous ethanol. Dissolve 1.91 g of sodium tetraborate, 0.1 g of SDS, and 88 mg of DTT in 50 mL of deionized water. Combine these two solutions and transfer to a 100 mL brown volumetric flask with deionized water to volume. Prepare a 0.1 mg / mL serine standard solution. Transfer 0, 100, 200, 300, and 400 μL of the serine standard solution to a 5 mL test tube. Fill the volume to 400 μL with deionized water. Add 3 mL of OPA reagent, mix, and react for 2 minutes. Measure the absorbance at 340 nm. Prepare a calibration curve for serine concentration and absorbance.

[0053] Dilute 1 mL of the enzymatic supernatant 50-fold. Mix 400 μL of the diluted sample, distilled water, and the serine standard solution with 3 mL of the LOP A reagent solution and place in a dark room for 2 minutes. Then, measure the absorbance at 340 nm using a spectrophotometer. Calculate the degree of hydrolysis using the following formula: , , , Among them, W S-NH2 is the amino content per gram of protein (mmol / g); C S-NH2 is the concentration of free amino groups in the diluent (mmol / L), which can be obtained from the standard curve according to the measured sample absorbance; V is the volume of the sample hydrolyzate (L); N is the dilution factor of the hydrolyzate; X is the sample mass (g); P is the mass fraction of protein in the sample (%); h is the number of peptide bonds broken per gram of protein (mmol / g); h tot is the total number of peptide bonds in each gram of egg yolk protein (mmol / g), which is taken as 8; α and β are constants, which are taken as 1 and 0.4 respectively.

[0054] ACE inhibitory activity was determined in boric acid-borax buffer (BBS buffer) (0.1 mol / L, pH 8.3, 0.3 mol / L NaCl). The initial sample solution (40 μL) and HHL (5 mM, 100 μL) were preincubated at 37°C for 5 minutes. Then, 10 μL of ACE (0.1 U / mL) was added, and the mixture was incubated at 37°C for 1 hour. HCl (250 μL, 1 M) and 1 mL of ethyl acetate were added, mixed by vortexing for 30 seconds, and centrifuged at 4000 rpm for 10 minutes. When layers were visible, 750 μL of the upper layer was transferred to a clean container, dried in a 110°C oven (approximately 15 minutes), and reconstituted with 3 mL of deionized water. After vortexing, the absorbance was measured at 228 nm. ACE inhibition was calculated using the following formula: , Wherein: A is the absorbance of the sample group (ACE and inhibitor) at 228 nm; B is the absorbance of the control group (ACE without inhibitor) at 228 nm; C is the absorbance of the blank group (without ACE and inhibitor) at 228 nm.

[0055] Solubility determination: Powder solubility was determined by placing 1 g of egg yolk powder in a 50 mL centrifuge tube and adding 30 mL of deionized water. Vortex the tube for 5 minutes. Then, centrifuge the tube at 3000 rpm for 10 minutes to collect the precipitate. Remove the tube, discard the supernatant, add 30 mL of deionized water, vortex the tube, and centrifuge again for 10 minutes. Dry the tube in a blast dryer for 8 hours and weigh it.

[0056] , Where m is the sample mass (g); m1 is the weight of the centrifuge tube + sediment after constant weight (g); and m2 is the mass of the centrifuge tube (g).

[0057] Protein solubility was determined by dissolving 200 mg of sample (protein content w2) in sodium phosphate buffer at pH 7.0 and stirring at 25°C for 30 minutes. The sample was then centrifuged at 5000 rpm for 10 minutes at 4°C. The soluble protein content w1 of the supernatant was determined using the Lowry method. Protein solubility was calculated using the following formula: .

[0058] Table 1 Preparation of single enzyme sample set

[0059] Single enzyme hydrolysis results: Figure 1 or Figure 2As shown in the figure, the DH and ACE inhibition rates of defatted egg yolk protein treated with different proteases differed significantly. The alkaline protease group had the highest DH, exceeding 35%, and the corresponding ACE inhibition rate was also high. Papain had relatively low DH and ACE inhibition rates, while the pepsin group had relatively low DH but a higher ACE inhibition rate, likely due to the production of more hydrophobic peptides.

[0060] The experimental results confirmed a certain correlation between DH and ACE inhibition. Based on this, alkaline protease was selected as the preferred enzyme to achieve higher DH and release hydrophobic active peptides, while other enzymes were used to increase peptide diversity.

[0061] The optimal single enzyme was screened based on the degree of hydrolysis and ACE inhibitory activity for dual-enzyme compound hydrolysis. Two dual-enzyme hydrolysis methods were set up: mixed and step-by-step. To ensure the total enzyme addition amount was 6000 U / g, the experimental method was designed as follows: 1) ANH: A mixture of 4000 U / g alkaline protease and 2000 U / g neutral protease, pH = 7.5, enzymatic hydrolysis temperature 55°C, enzymatic hydrolysis time 4 h.

[0062] 2) AH+NH: 3000 U / g alkaline protease was added for 2 h at pH 8 and then the enzyme was inactivated. 3000 U / g neutral protease was then added for 2 h at pH 7.

[0063] 3) NH+AH: 4000 U / g neutral protease was added for enzymatic hydrolysis at pH = 7 for 2 h, and then the enzyme was inactivated. Then 2000 U / g alkaline protease was added for enzymatic hydrolysis at pH = 8 for 2 h.

[0064] 4) AFH: a mixture of 3000 U / g alkaline protease and 3000 U / g flavor protease, pH = 7.5, enzymatic hydrolysis temperature 55°C, and enzymatic hydrolysis time 4 h.

[0065] 5) Example 2: AH+FH: 3000 U / g alkaline protease was enzymatically hydrolyzed at pH = 7 for 2 h, then the enzyme was inactivated. 3000 U / g flavor protease was then added and enzymatically hydrolyzed at pH = 7 for 2 h.

[0066] 6) Example 5: FH+AH: 3000 U / g flavor protease was enzymatically hydrolyzed at pH = 7 for 2 h, then the enzyme was inactivated. 3000 U / g alkaline protease was then added and enzymatically hydrolyzed at pH = 7.5 for 2 h.

[0067] Double enzyme compound enzymolysis results: The enzymatic solution pretreated with alkaline protease was compounded with flavor protease in steps (see Figure 3The results showed that the AH+FH dual enzyme step-by-step combination of Examples 2 and 3 significantly improved the degree of proteolysis and ACE inhibitory activity. The sequential action of alkaline protease and flavor protease increased DH from 35.20% of the single enzyme to 42.93%, and the ACE inhibition rate was also significantly improved ( Figure 4 ). In contrast, the simultaneous addition of enzymes or the addition of other enzymes alone (in order) were not as effective as 5) The step-by-step compounding of AH+FH showed good results, indicating that the step-by-step strategy of the present invention enables the enzymes to act more fully on different parts of the protein, thereby releasing more small molecule peptides. The resulting synthetic peptides KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY have ACE inhibitory and renin inhibitory activities, and have good ADMET properties.

[0068] The solubility of the dry powder sample and protein solubility of the enzymatic hydrolysate of the present invention are both maintained at a high level (see Figure 5 or Figure 6 ), compared with the control group, all enzymatic hydrolysis groups significantly increased the powder solubility and protein solubility of egg yolk powder. The solubility of the dual-enzyme hydrolysis was generally higher than that of the single enzyme. The dual enzyme has more recognition sites, enabling more extensive hydrolysis and promoting the release of small molecule peptides. The increase in protein solubility is mainly due to the enzymatic hydrolysis that frees more amino and carboxyl groups, allowing large insoluble aggregates to release more small soluble peptides. This indicates that the hydrolyzate obtained by dual-enzyme hydrolysis has advantages in water solubility and structural uniformity, which is beneficial for subsequent product preparation and activity retention.

[0069] 3. Preparation of ultrafiltration products The supernatant after enzymatic hydrolysis was subjected to peptide fractionation by ultrafiltration, and a total of 4 peptide fragments were fractionated: (1) The primary enzymatic hydrolysate is passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain a first filtrate with a molecular weight of less than 1 kDa, which is prepared into a freeze-dried powder for later use.

[0070] (2) The primary enzymatic hydrolysate is first passed through an ultrafiltration membrane with a pore size of 1 kDa, and the first retentate is then passed through an ultrafiltration membrane with a pore size of 5 kDa. The second filtrate is taken, and the molecular weight is 3 kDa ≤ ≤ 5 kDa, and is prepared into a freeze-dried powder for use.

[0071] (3) The primary enzymatic hydrolysate is first passed through an ultrafiltration membrane with a pore size of 5 kDa, and the first retentate is then passed through an ultrafiltration membrane with a pore size of 10 kDa. The second filtrate is taken, and the molecular weight is 5 kDa ≤ ≤ 10 kDa, and is prepared into a freeze-dried powder for use.

[0072] (4) The primary enzymatic hydrolysate is passed through an ultrafiltration membrane with a pore size of 10 kDa, and the second retentate with a molecular weight greater than 10 kDa is obtained and prepared into a freeze-dried powder for later use.

[0073] The ACE inhibitory activity of each fraction was determined ( Figure 7 ), it was found that the peptide fraction with a molecular weight <1 kDa had the highest inhibition rate, reaching 70.51%. The fraction <1 kDa was selected as the active enrichment for LC-MS / MS analysis.

[0074] Through mass spectrometry, 559 egg-derived peptides were identified. Source analysis showed that most of the active peptides were derived from PV protein in egg yolk, accounting for 39.4%, and 2-12 amino acids accounted for about 93% (such as Figure 8 or Figure 9 PeptideRanker was used to predict the potential activity of the peptides. Sequence alignment was performed through Biopep database analysis, resulting in the screening of 31 peptides. Hydrophobicity and toxicity analysis using ToxinPred revealed that all 31 peptides were non-toxic. Thirteen highly hydrophobic peptides were further screened for ADMET property analysis and activity prediction. The results showed that KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY exhibited ACE and renin inhibitory activities, as well as good ADMET properties.

[0075] Molecular docking analysis: After constructing the structural model of the five peptides screened, molecular docking simulation was performed with ACE protein ( Figure 10 ).

[0076] Through MOE molecular docking simulation, it was found that the peptides all showed stable binding ability to ACE, and their binding energies were all negative, indicating that this type of peptide formed a stable complex with ACE (Table 2). Among them, KFIPL and KFLPGY formed four hydrogen bonds with the key residue Glu384 of the S1 active pocket of ACE, and formed metal coordination or ionic bonds with the Zn701 ion, showing a mixed competitive inhibition mechanism similar to enalapril. KFLPTF formed five hydrogen bonds with inactive residues such as Glu123, Val399, Arg402, Ala356 and Arg522, and its mechanism of action was non-competitive inhibition. APTFAKL can form three hydrogen bonds with ACE, including binding to Glu411, which may interfere with Zn 2+ KLPDMILY forms up to eight hydrogen bonds with the lowest binding energy (−13.54 kcal / mol), including binding to the key action sites of Glu383 and Glu411, which can cause conformational changes in ACE and lead to its inactivation.

[0077] Table 2 Molecular docking analysis of peptides

[0078] The synthetic peptides KFIPL, KFLPGY, KFLPTF, APTFGKL and KLPDMILY were obtained by solid phase synthesis and prepared into lyophilized powder for use.

[0079] The synthetic peptides KFIPL, KFLPGY, KFLPTF, APTFGKL and KLPDMILY were tested for their ACE inhibitory activity in vitro ( Figure 11 KFLPGY, APTFGKL, and KLPDMILY exhibited the highest ACE inhibitory activities, at 91.57%, 92.79%, and 94.87%, respectively. KFLPTF, which exhibits non-competitive inhibition, exhibited lower ACE inhibitory activity of 60.97%. KFIPL, which exhibits mixed competitive inhibition, also exhibited high ACE inhibitory activity of 74.05%.

[0080] The present invention further utilizes the egg yolk hydrolysate containing the highly active peptide segments to prepare a functional beverage and conducts experiments.

[0081] All beverage samples were stirred for 30 minutes. The pH was adjusted with citric acid and sodium citrate. The mixture was pasteurized by storing it at 80°C for 10 minutes and then cooled in an ice bath for 5 minutes.

[0082] Zeta potential measurement: After the sample was diluted 50 times, the surface charge distribution of the droplets in the diluted sample was measured using a Nano-ZS laser light scattering instrument.

[0083] Particle Size Distribution: The emulsion particle size distribution was determined using a Mastrersizer 2000 laser particle size analyzer. The surface mean diameter (d32) and volume mean diameter (d43) were used to characterize the average particle size of the emulsion. A few drops of sample solution were slowly added to a beaker filled with distilled water using a rubber-tipped dropper. Sample testing began after the sample volume reached the set opacity range (10%-15%) and the opacity value stabilized.

[0084] Multiple light scattering analysis: A pulsed near-infrared light source (wavelength 850 nm) periodically scans the sample from top to bottom. Scans are performed over a 3-hour period with 3-minute intervals. The backscattered light intensity (ΔBS) and Turbiscan stability index (TSI) are collected using this instrument.

[0085] The resulting beverage samples were analyzed for particle size distribution, zeta potential, phase separation, turbidity, and microstructure. Zeta potential measured at different pH conditions showed that the absolute value of the zeta potential of the beverage system was the largest (negatively charged particles) at a pH of approximately 4, indicating the system was most stable. Macroscopic observations after 5 days of storage at 25°C ( Figure 12 ) showed that no obvious phase separation occurred in the beverages under the composite polysaccharide system, which was in sharp contrast to the control group without polysaccharide. Particle size test results ( Figure 13 ) showed that the average particle size of suspended particles in the beverage increased significantly after the addition of polysaccharides, but the distribution was narrow. For example, the d43 of the control group was only about 15.8μm, while after adding 0.45% CMC, the d43 increased to 57.4μm, and after adding 0.40% GG, it increased to 73.0μm; the d43 of the compound group (CMC 0.45% + GG 0.30%) was about 55.7μm. This shows that polysaccharides cause the peptides to aggregate into relatively large but stable composite particles, which is consistent with the system Zeta potential and other test results. In the backscattered light (ΔBS) analysis ( Figure 14 ), the ΔBS curve of the polysaccharide-added group samples was gentle, especially in the top and bottom areas, where the ΔBS change decreased, indicating that the sample sedimentation rate slowed down. By comparison, it can be seen that the ΔBS in the bottom area of ​​the control group increased significantly over time (particle sedimentation), while the ΔBS in the polysaccharide compound group remained almost stable. In addition, quantitative analysis using the total instability index (TSI) showed that the CMC+GG compound group had the lowest TSI and the highest stability. Microrheological test ( Figure 15 or Figure 16 or Figure 17 ) showed that beverages containing a composite polysaccharide system exhibited higher viscosity and elasticity indices at low shear rates, while the flow index decreased, indicating a more gel-like and viscous system, which contributes to the long-term stability of suspended particles. This demonstrates that beverages prepared in accordance with the present invention possess a uniform texture, high viscoelasticity, and excellent stability, and are less susceptible to sedimentation or stratification during pasteurization and subsequent storage.

[0086] In summary, the present invention not only solves the problem of polypeptide instability after sterilization in functional beverages, but also uses CMC and GG as stabilizers to solve the problem of enzymatic polypeptide utilization in functional beverages. It also adopts step-by-step enzymatic hydrolysis to prepare highly active ACE inhibitors, and uses bioinformatics methods with the help of an online active peptide database to screen out 5 new ACE inhibitory peptides from enzymatically hydrolyzed egg yolk, and elucidate the inhibition mechanism.

[0087] The above specific embodiments do not constitute a limitation on the scope of protection of this disclosure. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations, and substitutions may be made based on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure shall be included within the scope of protection of this disclosure.

Claims

1. A method for preparing an egg yolk polypeptide having ACE inhibitory activity, characterized in that: The method comprises: Q1. Dissolve defatted egg yolk powder in water and adjust the pH to 6-8. Q2. Add alkaline protease or flavor protease and perform enzymatic hydrolysis at 50-60°C and pH 7-8 for 1.5-2.5 hours, then inactivate the enzyme to obtain egg yolk hydrolyzate. Q3. Add flavor protease or alkaline protease to the egg yolk hydrolyzate, continue enzymatic hydrolysis at 50-60°C, pH = 7-8 for 1.5-2.5h, and then inactivate the enzyme to obtain an egg yolk enzymatic hydrolyzate; Q4. freeze-drying the egg yolk enzymatic hydrolysate to obtain egg yolk polypeptide.

2. The method for preparing an egg yolk polypeptide having ACE inhibitory activity according to claim 1, wherein: The dosage of the alkaline protease is 2000-4000 U / g.

3. The method for preparing an egg yolk polypeptide having ACE inhibitory activity according to claim 1, wherein: The amount of the flavor protease is 2000-4000 U / g.

4. The method for preparing an egg yolk polypeptide having ACE inhibitory activity according to claim 1, wherein: The egg yolk enzymatic hydrolysate was passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain a first filtrate with a molecular weight of less than 1 kDa, which was prepared into a freeze-dried powder for later use.

5. An egg yolk polypeptide prepared by the method of claim 4, characterized in that: The amino acid sequence of the egg yolk polypeptide is any one or more combinations of the following: KFIPL, KFLPGY, KFLPTF, APTFGKL, KLPDMILY.

6. Use of the egg yolk polypeptide prepared by the method according to any one of claims 1 to 5 having ACE inhibitory activity in functional active beverages.

7. A functional beverage with ACE inhibitory activity containing the egg yolk polypeptide according to any one of claims 1 to 5, characterized in that: The pH of the egg yolk enzymatic hydrolysate is adjusted to 3.5-5.0 with a citric acid / sodium citrate buffer system, and the egg yolk enzymatic hydrolysate is pasteurized at 80° C. for 10 minutes and rapidly cooled to obtain a functional beverage with ACE inhibitory activity.

8. The functional beverage having ACE inhibitory activity according to claim 7, characterized in that: It also includes adding 3% orange juice powder based on the mass ratio of egg yolk enzymatic hydrolysate.

9. The functional beverage having ACE inhibitory activity according to claim 7, characterized in that: Two polysaccharides, carboxymethyl cellulose (CMC) and guar gum (GG), are also included as stabilizers.

10. The functional beverage having ACE inhibitory activity according to claim 9, characterized in that: The carboxymethyl cellulose (CMC) accounts for 0.45% of the mass of the egg yolk enzymolyte, and the guar gum accounts for 0.30% of the mass of the egg yolk enzymolyte.

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