Preparation method and application of egg yolk polypeptide with ACE inhibitory activity

Abstract

The present application relates to a kind of preparation method and application of egg yolk polypeptide with ACE inhibitory activity, the method comprises: Q1.degreased egg yolk powder is dissolved in appropriate amount of water, adjust pH to 6~8;Q2.50~60 ℃, pH=7‑8 under the condition of adding alkaline protease or flavour protease is carried out enzymolysis 1.5~2.5h, then enzyme is killed, and the egg yolk hydrolysate is obtained.The present application not only uses step-by-step enzymatic hydrolysis to prepare high-activity ACE inhibitor, and using the method of bioinformatics, 5 ACE inhibitory peptides (KFIPL, KFLPGY, KFLPTF, ATPFGKL, KLPDMILY) with high activity are screened and identified, and in order to solve the problem of polypeptide instability after sterilization in functional beverage, CMC and GG are used as stabilizer, and the problem of enzymatic polypeptide in functional beverage is solved.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a method for preparing and applying an egg yolk polypeptide with ACE inhibitory activity. Background Technology

[0002] Hypertension poses an increasingly serious threat to human health, being a major risk factor for myocardial infarction, heart failure, kidney failure, stroke, and even death. Angiotensin-converting enzyme (ACE) is a key enzyme in blood pressure regulation, catalyzing the conversion of angiotensin I into the highly reactive angiotensin II, one of the strongest vasoconstrictors. This vasoconstriction reduces blood vessel diameter and increases peripheral resistance, thereby raising 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 often results in adverse reactions such as cough, headache, and taste disturbances. Therefore, developing safe, non-toxic, and widely available natural ACE inhibitors has become a research hotspot in the fields of functional foods and food-medicine homology in recent years.

[0003] Eggs contain all the nutrients necessary for embryonic development, with a protein bioavailability rate as high as 98%. Egg yolks, as a nutrient-rich food, are high in protein and have attracted increasing attention in recent years in bioactive peptide research. Egg yolk protein is mainly composed of vitellin (38%, in α, β, and γ forms), vitellin lipoprotein (36%, in α and β forms), low-density lipoprotein (17%), and PV (8-9%). Existing research shows that egg yolk protein has high bioavailability and a unique amino acid composition, especially PV protein and low-density lipoprotein, which have great potential in the extraction of bioactive peptides. Therefore, developing safe and efficient food-derived hypotensive peptides from egg yolks, and subsequently developing functional foods, is crucial for increasing the utilization rate of egg yolk resources and balancing the development of the egg product industry.

[0004] However, traditional enzymatic methods for preparing peptides suffer from limitations such as limited enzyme selection and low functional peptide release efficiency, making it difficult to obtain highly active target peptides. Furthermore, the resulting peptides exhibit poor stability during processing, particularly under conditions like high temperatures and pasteurization, leading to precipitation, turbidity, and reduced functional activity, thus limiting their widespread application in liquid foods such as beverages. In addition, the lack of systematic screening and validation of the relationship between functional peptide structure and activity results in unstable functional effects of the developed products, hindering industrialization. Therefore, there is an urgent need to develop an efficient peptide preparation and purification strategy, combined with food formulation design, to improve its stability and practicality in liquid products. Summary of the Invention

[0005] In view of the above problems, this invention provides a method for preparing egg yolk peptides with ACE inhibitory activity and their application. This method not only addresses the problem of peptide instability after sterilization in functional beverages by using CMC and GG as stabilizers to solve the problem of utilizing enzymatically hydrolyzed peptides in functional beverages, but also employs stepwise enzymatic hydrolysis to prepare highly active ACE inhibitors. Furthermore, using bioinformatics methods and an online active peptide database, five new ACE inhibitory peptides were screened from enzymatically hydrolyzed egg yolks, and the inhibition mechanism was elucidated.

[0006] To achieve the above and other related objectives, the present invention provides the following technical solution: a method for preparing an egg yolk polypeptide with ACE inhibitory activity, the method comprising:

[0007] Q1. Dissolve the defatted egg yolk powder in an appropriate amount of water and adjust the pH to 6-8;

[0008] Q2. Add alkaline protease or flavor protease at 50-60℃ and pH=7-8 for 1.5-2.5 hours, then inactivate the enzyme to obtain egg yolk hydrolysate;

[0009] Q3. Add flavor protease or alkaline protease to the egg yolk hydrolysate, and continue enzymatic hydrolysis for 1.5 to 2.5 hours at 50-60°C and pH 7-8, then inactivate the enzyme to obtain egg yolk hydrolysate;

[0010] Q4. Freeze-dry the egg yolk hydrolysate to obtain egg yolk polypeptide.

[0011] Preferably, the amount of alkaline protease used is 2000-4000 U / g.

[0012] Preferably, the amount of the flavor protease used is 2000-4000 U / g.

[0013] Preferably, the egg yolk hydrolysate 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 then freeze-dried into a powder for later use.

[0014] 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.

[0015] A peptide, wherein the peptide is used to prepare an ACE-inhibiting drug or a food with an auxiliary ACE-inhibiting function, or to determine the ACE-inhibiting effect for purposes other than disease diagnosis and treatment, wherein the amino acid sequence of the peptide is any one or more of the following: KFIPL, KFLPGY, KFLPTF, APTFGKL, KLPDMILY.

[0016] The egg yolk peptides prepared by the above method have ACE inhibitory and renin inhibitory activities and can be used in functional active beverages.

[0017] A functional beverage with ACE inhibitory activity is prepared by adjusting the pH of the egg yolk hydrolysate to 3.5–5.0 using a citric acid / sodium citrate buffer system, pasteurizing at 80°C for 10 minutes, and then rapidly cooling. The beverage also includes 3% orange juice powder, carboxymethyl cellulose (CMC), and guar gum (GG) as stabilizers, added according to the mass ratio of the egg yolk hydrolysate. The carboxymethyl cellulose (CMC) accounts for 0.45% of the mass of the egg yolk hydrolysate, and the guar gum accounts for 0.30% of the mass of the egg yolk hydrolysate.

[0018] The present invention has the following positive effects:

[0019] 1. This invention significantly improves the release efficiency of ACE-inhibiting active substances by optimizing the protease and employing a dual-enzyme synergistic hydrolysis technique. 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.

[0020] 2. This invention successfully screened multiple small molecule peptides (KFIPL, KFLPGY, KFLPTF, APTFGKL, KLPDMILY) with high ACE inhibitory activity through ultrafiltration purification and peptide screening technology, and precisely located their mechanisms of action. The screened functional peptides possess high biological activity, low toxicity, and favorable pharmacokinetic properties, and have the potential to be used as functional factors in the development of antihypertensive foods or related formulations.

[0021] 3. This invention effectively improves the stability of functional polypeptide beverages during pasteurization through a polysaccharide complex system. The selected polysaccharides and polypeptides have good charge complementarity and interaction capabilities, which can enhance the structural stability of the system at the molecular level, extend the product shelf life, and improve the overall quality and functional retention of the beverage. Attached Figure Description

[0022] Figure 1 A schematic diagram of (A)DH, the single-enzyme hydrolysate of the present invention;

[0023] Figure 2 This is a schematic diagram illustrating the (B) ACE inhibitory activity of the single-enzyme hydrolysate of the present invention;

[0024] Figure 3 A schematic diagram of (A)DH of the dual-enzyme hydrolysate of the present invention;

[0025] Figure 4This is a schematic diagram illustrating the (B) ACE inhibitory activity of the dual-enzyme hydrolysate of the present invention;

[0026] Figure 5 This is a schematic diagram illustrating the solubility of different enzymatic hydrolysate powders of the present invention;

[0027] Figure 6 This is a schematic diagram illustrating the protein solubility of the present invention;

[0028] Figure 7 This is a schematic diagram illustrating the ACE inhibitory activity of the ultrafiltration components of the present invention;

[0029] Figure 8 This is a schematic diagram illustrating the sequencing results analysis of the polypeptide source (A) of the present invention;

[0030] Figure 9 This is a schematic diagram of the sequencing results analysis of the polypeptide length distribution of the present invention (B);

[0031] Figure 10 The diagram shows the molecular docking of the five active ACE-inhibiting peptides of this invention.

[0032] Figure 11 The ACE inhibitory activity of the synthesized peptide;

[0033] 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.

[0034] Figure 13 The images show the particle size distribution and macroscopic images of different polysaccharide-stabilized beverages of the present invention. (A) Particle size distribution, (B) Macroscopic image at 25°C.

[0035] Figure 14 A schematic diagram showing the ΔBS values ​​of polysaccharide-stabilized beverages with different formulations according to the present invention;

[0036] Figure 15 (A) Schematic diagram of the macroscopic viscosity index of the polysaccharide stable beverages with different ratios according to the present invention;

[0037] Figure 16 This is a schematic diagram of the elastic index (B) of the microrheological analysis of polysaccharide-stabilized beverages with different ratios according to the present invention.

[0038] Figure 17 This is a schematic diagram of the flowability index of the polysaccharide stable beverages with different ratios according to the present invention, based on microrheological analysis (C). Detailed Implementation

[0039] The exemplary embodiments of this disclosure are described below with reference to the accompanying drawings, including various details of the embodiments to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this disclosure. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0040] Example 1: Preparation of egg yolk peptides and screening of target peptides.

[0041] (1) Preparation of defatted egg yolk powder: Egg yolk powder and ethyl acetate were diluted at a ratio of 1:4 (egg yolk powder g: ethyl acetate mL). The ultrasonic temperature was controlled at 0 ℃, the ultrasonic power at 100 W, and the ultrasonic treatment time at 15 min (ultrasound 3 s, stop 3 s). All ultrasonically treated samples were filtered through a G3 Buchner funnel, and the filter cake was placed in a vacuum drying oven at 40 ℃ to evaporate the solvent for about 24 h. After that, it was pulverized to obtain defatted egg yolk powder for the next step.

[0042] (2) Enzymatic hydrolysis: The egg yolk powder obtained in step (1) was dispersed in deionized water to prepare a suspension with a mass fraction of 5% (egg yolk powder / water, w / w). Enzymatic hydrolysis was performed using 4000 U / g alkaline protease at pH=8 and 60℃ for 2.5 h. After enzyme inactivation, the egg yolk hydrolysate was obtained. Then, 2000 U / g flavor protease was used for enzymatic hydrolysis at pH=8 and 55℃ for 1.5 h. After hydrolysis, the hydrolysate was placed in a boiling water bath for 5 min to inactivate the enzyme, and then cooled to obtain the egg yolk hydrolysate for later use. The egg yolk hydrolysate was passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain the first filtrate, with a molecular weight <1 kDa, which was then freeze-dried into a powder for later use.

[0043] (3) Screening and identification of target peptides

[0044] The egg yolk peptides obtained in step (2) were fractionated by ultrafiltration, resulting in four peptide fragments. Peptides <1 kDa showed the highest activity. Further mass spectrometry identification revealed 559 egg-derived peptides. Source analysis indicated that most active peptides originated from PV proteins in the egg yolk, accounting for 39.4%. Peptide Ranker was used to predict the potential activity of the peptides. Sequence alignment using the Biopep database identified 31 peptides. ToxinPred analysis of the peptides' hydrophobicity and toxicity showed that none of the 31 peptides were toxic. Thirteen highly hydrophobic peptides were selected for ADMET property analysis and activity prediction. The results showed that KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY exhibited ACE inhibitory and renin inhibitory activities, and good ADMET properties. Molecular docking was then performed on these five peptides. The results showed that KFIPL and KFLPGY may interact with ACE through mixed competitive inhibition, while KFLPTF, APTFGKL, and KLPDMILY interact with ACE through non-competitive inhibition. All five peptides could interact with Zn 701 via metal coordination or ionic bonding. Among them, KLPDMILY formed 8 hydrogen bonds with ACE, with the lowest binding energy of -13.539 kcal / mol.

[0045] Example 2: Preparation of egg yolk peptides and screening of target peptides.

[0046] (1) Preparation of defatted egg yolk powder: Egg yolk powder and ethyl acetate were diluted at a ratio of 1:6 (egg yolk powder g: ethyl acetate mL). The ultrasonic temperature was controlled at 3℃, the ultrasonic power at 200 W, and the ultrasonic treatment time at 25 min (ultrasound 3 s, stop 3 s). All ultrasonically treated samples were filtered through a G3 Buchner funnel, and the filter cake was placed in a vacuum drying oven at 40℃ to evaporate the solvent for about 24 h. After that, it was pulverized to obtain defatted egg yolk powder for the next step.

[0047] (2) Enzymatic hydrolysis: The egg yolk powder obtained in step (1) was dispersed in deionized water to prepare a suspension with a mass fraction of 6% (egg yolk powder / water, w / w). Enzymatic hydrolysis was performed using 3000 U / g alkaline protease at pH 7 and 50℃ for 2 hours to obtain the egg yolk hydrolysate. After hydrolysis, the enzyme was inactivated, and then 3000 U / g flavor protease was used for enzymatic hydrolysis at pH 7 and 55℃ for 2 hours. After hydrolysis, the hydrolysate was placed in a boiling water bath for 5 minutes to inactivate the enzyme, and then cooled to obtain the egg yolk hydrolysate for later use. The egg yolk hydrolysate was passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain the first filtrate, with a molecular weight < 1 kDa, which was then freeze-dried into a powder for later use.

[0048] (3) Screening and identification of target peptides.

[0049] The egg yolk polypeptide obtained in step (2) was fractionated by ultrafiltration, resulting in four polypeptide fragments. Among them, the polypeptide with <1kDa had the highest activity.

[0050] Example 3: Preparation of egg yolk peptides and screening of target peptides.

[0051] The difference from Example 2 is:

[0052] (2) Enzymatic hydrolysis: The egg yolk powder obtained in step (1) was dispersed in deionized water to prepare a suspension with a mass fraction of 6% (egg yolk powder / water, w / w). Enzymatic hydrolysis was performed using 2000 U / g alkaline protease at pH=7 and 53℃ for 2 hours. After enzyme inactivation, the egg yolk hydrolysate was obtained. Then, 4000 U / g flavor protease was used for enzymatic hydrolysis at pH=7.5 and 57℃ for 2 hours. After hydrolysis, the hydrolysate was placed in a boiling water bath for 5 minutes to inactivate the enzyme, and then cooled to obtain the egg yolk hydrolysate for later use. The egg yolk hydrolysate was passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain the first filtrate, with a molecular weight <1 kDa, which was then freeze-dried into a powder for later use.

[0053] Example 4: Preparation of egg yolk peptides and screening of target peptides.

[0054] (1) Preparation of defatted egg yolk powder.

[0055] Egg yolk powder and ethyl acetate were diluted at a ratio of 1:8 (egg yolk powder g: ethyl acetate mL). The ultrasonic temperature was controlled at 4℃, the ultrasonic power at 400 W, and the ultrasonic treatment time at 10 min (3 s ultrasonic waves followed by 3 s pause). All ultrasonically treated samples were filtered through a G3 Buchner funnel, and the filter cake was placed in a vacuum drying oven at 40℃ to evaporate the solvent for about 24 h. After evaporation, the filtered cake was pulverized to obtain defatted egg yolk powder for further use.

[0056] (2) Enzymatic hydrolysis of defatted egg yolk powder.

[0057] The egg yolk hydrolysate was obtained by enzymatic hydrolysis using 2000 U / g flavor protease at pH 8 and 55°C for 2 hours, followed by enzyme inactivation. Then, the hydrolysate was hydrolyzed using 4000 U / g alkaline protease at pH 7.5 and 52°C for 1.5 hours. After hydrolysis, the hydrolysate was placed in a 100°C water bath for 10 minutes to inactivate the enzyme, yielding the egg yolk hydrolysate. The egg yolk hydrolysate was passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain the first filtrate, with a molecular weight <1 kDa, which was then freeze-dried into a powder for later use.

[0058] (3) Screening, identification and synthesis of target peptides

[0059] Ultrafiltration products with molecular weight <1 kDa were subjected to HPLC-MS / MS. The mobile phases for HPLC included mobile phase A and mobile phase B; the elution column was a domestically produced fused silica capillary column (ID 75 µm, 150 mm, Upchurch, Oak Harbor, WA) containing C-18 resin (300 Å, 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: 0–8 min 3% B, 8–40 min 3–50% B, 40–55 min 50% B, 55–75 min 50–99% B, 75–85 min 99–3% B; the flow rate was 0.3 μL / min; and the injection volume was 1 μL.

[0060] Source analysis shows that most of the bioactive peptides originate from PV protein in egg yolk, accounting for 39.4%, with amino acids 2-12 accounting for approximately 93% (e.g., ...). Figure 8 or Figure 9 (As shown).

[0061] The synthetic peptides KFIPL, KFLPGY, KFLPTF, APTFGKL and KLPDMILY obtained by solid-phase synthesis were prepared into lyophilized powders for later use.

[0062] Example 5: Preparation of egg yolk peptides and screening of target peptides.

[0063] The difference from Example 3 is:

[0064] (2) Enzymatic hydrolysis of defatted egg yolk powder.

[0065] The egg yolk hydrolysate was obtained by enzymatic hydrolysis using 3000 U / g flavor protease at pH 7 and 50°C for 2 hours, followed by enzyme inactivation. Then, the hydrolysate was further hydrolyzed using 3000 U / g alkaline protease at pH 7.5 and 55°C for 2 hours. After hydrolysis, the hydrolysate was placed in a 100°C water bath for 10 minutes to inactivate the enzyme, yielding the egg yolk hydrolysate. The egg yolk hydrolysate was then passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain the first filtrate, with a molecular weight <1 kDa, which was then freeze-dried into a powder for later use.

[0066] Example 6: Preparation of egg yolk peptides and screening of target peptides.

[0067] The difference from Example 3 is:

[0068] (2) Enzymatic hydrolysis of defatted egg yolk powder.

[0069] The egg yolk hydrolysate 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. Then, the hydrolysate was further hydrolyzed using 2000 U / g alkaline protease at pH 7.5 and 60°C for 2 hours. After hydrolysis, the hydrolysate was placed in a 100°C water bath for 10 minutes to inactivate the enzyme, yielding the egg yolk hydrolysate. The egg yolk hydrolysate was then passed through an ultrafiltration membrane with a pore size of 1 kDa to obtain the first filtrate, which had a molecular weight <1 kDa and was freeze-dried into a powder for later use.

[0070] Example 7: Preparation of a functional beverage with ACE inhibition. This invention further utilizes the above-mentioned enzymatic hydrolysate containing highly active peptides to prepare a functional beverage.

[0071] 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 to add different concentrations of CMC and GG to the mixture.

[0072] CMC was added at concentrations of 0.3% (0.3 parts, 0.3% of egg yolk 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".

[0073] 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”.

[0074] A mixture of 0.3% CMC and 0.2% GG was added and named "CMC+GG". The mixture without added polysaccharides served as a control and was named "control".

[0075] All samples were stirred for 30 min. The pH was adjusted with citric acid and sodium citrate. The mixtures were pasteurized at 80 °C for 10 min and then cooled by incubation in an ice bath for 5 min.

[0076] In the optimized formulation, 3% orange juice powder was added to the enzymatically hydrolyzed peptide solution by mass ratio to improve taste, and carboxymethyl cellulose (CMC) and guar gum (GG) were added as stabilizers. The optimal blending ratio was determined through comparative experiments; for example, a CMC ratio of 0.45% and a GG ratio of 0.30% resulted in optimal stability. After thoroughly mixing the above solution, the pH was adjusted to 3.5-5.0 using a citrate-sodium citrate buffer solution. Then, pasteurization was performed by heating in a water bath at 80°C for 10 minutes, followed by rapid cooling to obtain a stable beverage sample.

[0077] Experimental example:

[0078] The testing methods for relevant data in embodiments of the present invention are as follows:

[0079] Determination of hydrolysis degree: In the dark, protected from light, dissolve 80 mg of OPA in 2 mL of anhydrous ethanol. Then dissolve 1.91 g of sodium tetraborate, 0.1 g of SDS, and 88 mg of DTT in 50 mL of deionized water. Mix these two solutions and transfer to a 100 mL amber volumetric flask with deionized water, making up to volume. Prepare a 0.1 mg / mL serine standard solution. Take 0, 100, 200, 300, and 400 μL of the serine standard solution into 5 mL test tubes, add deionized water to a final volume of 400 μL, add 3 mL of OPA reagent, mix well, react for 2 min, and then measure the absorbance at 340 nm. Plot a standard curve of serine concentration versus absorbance.

[0080] Take 1 mL of the supernatant after enzymatic hydrolysis and dilute it 50-fold. Mix 400 μL of the diluted sample, distilled water, and serine standard solution with 3 mL of LOPA reagent solution, and then place in a dark room for 2 min. Afterward, measure the absorbance at 340 nm using a spectrophotometer. The degree of hydrolysis is calculated using the following formula:

[0081] ,

[0082] ,

[0083] ,

[0084] Among them, W S-NH2 The amino group content per gram of protein (mmol / g); C S-NH2 V represents the concentration of free amino groups in the diluent (mmol / L), which is obtained from the standard curve based on the measured absorbance of the sample; V is the volume of the sample hydrolysate (L); N is the dilution factor of the hydrolysate; X is the sample mass (g); P is the protein mass fraction in the sample (%); h is the number of peptide bonds broken per gram of protein (mmol / g); h tot The total number of peptide bonds per gram of egg yolk protein (mmol / g) is 8; α and β are constants, taken as 1 and 0.4 respectively.

[0085] Assay for ACE inhibitory activity: The reaction was performed in borate-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 pre-incubated at 37 °C for 5 min. Then, 10 μL of ACE (0.1 U / mL) was added, and the mixture was further incubated at 37 °C for 1 h. HCl (250 μL, 1 M) and 1 mL of ethyl acetate were added, the mixture was shaken for 30 s, and centrifuged at 4000 rpm for 10 min. Layering was observed. 750 μL of the supernatant was transferred to a clean container, dried in a 110 °C oven (approximately 15 min), and then reconstituted with 3 mL of deionized water. After vortexing, the absorbance was measured at 228 nm. The ACE inhibition rate was calculated using the following formula:

[0086] ,

[0087] In the formula: 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.

[0088] Solubility determination: To determine the solubility of the powder, 1 g of egg yolk powder was placed in a 50 mL centrifuge tube, and 30 mL of deionized water was added. The tube was vortexed for 5 min. Then, it was centrifuged at 3000 rpm for 10 min, and the precipitate was collected. After removing the precipitate, the supernatant was removed, 30 mL of deionized water was added, the tube was shaken up and down, and centrifuged again for 10 min. The precipitate was then dried in a forced-air dryer for 8 h, and weighed.

[0089] ,

[0090] Where m is the sample mass (g); m1 is the weight of the centrifuge tube plus the precipitate after constant weight (g); and m2 is the centrifuge tube mass (g).

[0091] 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 min. The sample was then centrifuged at 5000 rpm for 10 min 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:

[0092] .

[0093] Table 1 Preparation of single enzyme sample groups

[0094]

[0095] Single-enzyme digestion results: such as Figure 1 or Figure 2 As shown, the inhibition rates of DH and ACE in defatted egg yolk protein treated with different proteases varied significantly. The alkaline protease group had the highest DH, exceeding 35%, and its corresponding ACE inhibition rate was also among the highest. Papain had relatively low DH and ACE inhibition rates, while the pepsin group had a relatively low DH but a high ACE inhibition rate, possibly due to the production of more hydrophobic peptides.

[0096] Experimental results confirmed a certain correlation between DH and ACE inhibition. Based on this, alkaline protease was selected as the preferred enzyme to obtain higher DH and release hydrophobic active peptides, while other enzymes were also used to increase peptide diversity.

[0097] The optimal single enzyme was screened based on its degree of hydrolysis and ACE inhibitory activity for dual-enzyme digestion. Two dual-enzyme digestion methods were set up: mixed digestion and stepwise digestion. To ensure a total enzyme addition of 6000 U / g, the experimental procedure was designed as follows:

[0098] 1) ANH: 4000 U / g alkaline protease and 2000 U / g neutral protease are mixed, pH=7.5, enzymatic hydrolysis temperature is 55℃, and enzymatic hydrolysis time is 4h.

[0099] 2) AH+NH: 3000U / g alkaline protease was hydrolyzed at pH=8 for 2 hours to inactivate the enzyme, and then 3000U / g neutral protease was added and hydrolyzed at pH=7 for 2 hours.

[0100] 3) NH+AH: 4000 U / g neutral protease was hydrolyzed at pH=7 for 2 hours to inactivate the enzyme, and then 2000 U / g alkaline protease was added and hydrolyzed at pH=8 for 2 hours.

[0101] 4) AFH: 3000 U / g alkaline protease and 3000 U / g flavor protease mixed, pH=7.5, hydrolysis temperature 55℃, hydrolysis time 4h.

[0102] 5) Example 2: AH+FH: 3000U / g alkaline protease was hydrolyzed at pH=7 for 2 hours and then the enzyme was inactivated. Then 3000U / g flavor protease was added and hydrolyzed at pH=7 for 2 hours.

[0103] 6) Example 5: FH+AH: 3000U / g flavor protease was enzymatically hydrolyzed at pH=7 for 2 hours and then inactivated. Then 3000U / g alkaline protease was added and enzymatically hydrolyzed at pH=7.5 for 2 hours.

[0104] Results of dual-enzyme hydrolysis: The hydrolysate pretreated with alkaline protease was combined with flavor protease for stepwise hydrolysis (see...). Figure 3The results showed that the stepwise combination of AH+FH dual enzymes in Examples 2 and 3 significantly improved the degree of proteolysis and ACE inhibition activity. Sequential action of alkaline protease and flavor protease increased the DH content from 35.20% to 42.93% with the single enzyme, and also significantly improved the ACE inhibition rate. Figure 4 In comparison, the simultaneous addition of enzymes or the addition of other enzymes individually (in order) did not achieve the same effect as the stepwise combination of AH+FH, indicating that the stepwise strategy of this invention enables the enzymes to act more fully on different parts of the protein, thereby releasing more small peptides. The resulting synthetic peptides KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY have ACE inhibitory and renin inhibitory activities, and also exhibit good ADMET properties.

[0105] The solubility of the dry powder sample and the protein solubility of the enzymatic hydrolysis product of this invention remain at a high level (see...). Figure 5 or Figure 6 Compared to the control group, all enzymatic hydrolysis groups showed significant improvements in the powder solubility of egg yolk powder and the solubility of egg white protein. The solubility of the dual-enzyme hydrolysis was generally higher than that of the single-enzyme hydrolysis. Dual enzymes have more recognition sites, enabling more extensive hydrolysis and promoting the release of small peptides. The increased protein solubility is mainly due to the enzymatic hydrolysis releasing more amino and carboxyl groups, allowing larger insoluble aggregates to release more small soluble peptides. This indicates that the hydrolysates obtained through dual-enzyme hydrolysis have advantages in water solubility and structural uniformity, which is beneficial for subsequent product preparation and activity maintenance.

[0106] 3. Preparation of ultrafiltration products

[0107] The supernatant after enzymatic hydrolysis was fractionated into peptides using ultrafiltration, yielding a total of four peptide fragments:

[0108] (1) Pass the primary enzymatic hydrolysate through an ultrafiltration membrane with a pore size of 1 kDa to obtain the first filtrate with a molecular weight of <1 kDa, and prepare it into a freeze-dried powder for later use.

[0109] (2) Pass the primary enzymatic hydrolysate through an ultrafiltration membrane with a pore size of 1 kDa, take the first retentate and then pass it through an ultrafiltration membrane with a pore size of 5 kDa, take the second filtrate, 3 kDa ≤ molecular weight ≤ 5 kDa, and prepare it into a freeze-dried powder for later use.

[0110] (3) Pass the primary enzymatic hydrolysate through an ultrafiltration membrane with a pore size of 5 kDa, take the first retentate and then pass it through an ultrafiltration membrane with a pore size of 10 kDa, take the second filtrate, 5 kDa ≤ molecular weight ≤ 10 kDa, and prepare it into a freeze-dried powder for later use.

[0111] (4) Pass the primary enzymatic hydrolysate through an ultrafiltration membrane with a pore size of 10 kDa, take the second retentate with a molecular weight > 10 kDa, and prepare it into a freeze-dried powder for later use.

[0112] ACE inhibitory activity was determined in each fraction of the distillate. Figure 7 The study found that peptides with a molecular weight <1 kDa exhibited the highest inhibition rate, reaching 70.51%. Peptides with a molecular weight <1 kDa were selected as the active concentrate for LC-MS / MS analysis.

[0113] Mass spectrometry identification revealed 559 egg-derived peptides. Source analysis showed that most of the bioactive peptides originated from PV protein in the egg yolk, accounting for 39.4%, with amino acids 2-12 comprising approximately 93% (e.g., ...). Figure 8 or Figure 9 (As shown). PeptideRanker was used to predict the potential activity of peptides. Sequence alignment analysis using the Biopep database identified 31 peptides. Hydrophobicity and toxicity analysis of the peptides using ToxinPred showed that none of the 31 peptides were toxic. Further screening identified 13 peptides with high hydrophobicity for ADMET property analysis and activity prediction. The results showed that KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY possessed ACE inhibitory and renin inhibitory activities, and exhibited good ADMET properties.

[0114] Molecular docking analysis: After constructing structural models of the five screened peptides, molecular docking simulations were performed with ACE proteins. Figure 10 ).

[0115] MOE molecular docking simulations revealed that all the peptides exhibited stable binding ability to ACE, with negative binding energies, indicating that these peptides form stable complexes with ACE (Table 2). Specifically, KFIPL and KFLPGY form four hydrogen bonds with Glu384, a key residue in the S1 active pocket of ACE, and form metal coordination or ionic bonds with Zn701 ions, exhibiting a mixed competitive inhibition mechanism similar to enalapril. KFLPTF forms five hydrogen bonds with inactive residues such as Glu123, Val399, Arg402, Ala356, and Arg522, with a non-competitive inhibition mechanism. APTFGKL can form three hydrogen bonds with ACE, including binding to Glu411, possibly by interfering with Zn701. 2+ The coordination structure plays an inhibitory role. KLPDMILY forms up to 8 hydrogen bonds with the lowest binding energy (-13.54 kcal / mol), including binding to key binding sites of Glu383 and Glu411, which can cause conformational changes in ACE, leading to its inactivation.

[0116] Table 2 Molecular docking analysis of peptides

[0117]

[0118] The synthetic peptides KFIPL, KFLPGY, KFLPTF, APTFGKL and KLPDMILY obtained by solid-phase synthesis were prepared into lyophilized powders for later use.

[0119] The synthetic peptides KFIPL, KFLPGY, KFLPTF, APTFGKL, and KLPDMILY were tested for their in vitro ACE inhibitory activity. Figure 11 KFLPGY, APTFGKL, and KLPDMILY showed the highest ACE inhibitory activity, at 91.57%, 92.79%, and 94.87%, respectively. KFLPTF, with non-competitive inhibition, showed lower ACE inhibitory activity at 60.97%. KFIPL, with mixed competitive inhibition, also showed high ACE inhibitory activity, reaching 74.05%.

[0120] The present invention further utilizes the egg yolk hydrolysate containing highly active peptides to prepare functional beverages and conducts experiments.

[0121] All beverage samples were stirred for 30 min. The pH was adjusted with citric acid and sodium citrate. The mixture was pasteurized at 80°C for 10 min and then cooled in an ice bath for 5 min.

[0122] Zeta potential determination: After diluting the sample 50 times, the surface charge distribution of the droplets in the diluted sample was determined using a Nano-ZS laser light scattering instrument.

[0123] Particle size distribution determination: The particle size distribution of the emulsion 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 sample beaker containing distilled water using a dropper. Once the sample volume reached the set occlusion range (10%-15%) and the occlusion value stabilized, sample testing began.

[0124] Multiple light scattering analysis: The sample was periodically scanned from top to bottom using a pulsed near-infrared light source (wavelength 850 nm). The scanning time was 3 hours, with 3-minute intervals. The back-scattered light intensity (ΔBS) and Turbiscan stability index (TSI) were collected using this device.

[0125] The obtained beverage samples were analyzed for particle size distribution, zeta potential, phase separation, turbidity, and microstructure. Zeta potentials measured under different pH conditions showed that the absolute value of the zeta potential was highest at approximately pH 4 (particles carrying a negative charge), indicating the most stable system. Macroscopic observations were performed after storage at 25℃ for 5 days. Figure 12 The results showed that no significant phase separation occurred in the beverages under the compound polysaccharide system, in stark contrast to the control group without added polysaccharides. Particle size test results ( Figure 13 The results showed that the average particle size of suspended particles in the beverage significantly increased but the distribution narrowed after the addition of polysaccharides. For example, the d43 of the control group was only about 15.8 μm, while it increased to 57.4 μm after adding 0.45% CMC and to 73.0 μm after adding 0.40% GG; the d43 of the compound group (0.45% CMC + 0.30% GG) was about 55.7 μm. This indicates that polysaccharides aggregate peptides into relatively large but stable composite particles, consistent with the system's zeta potential and other test results. In backscattered light (ΔBS) analysis ( Figure 14 The ΔBS change curve of the polysaccharide-added group was relatively flat, especially with a decrease in ΔBS change in the top and bottom regions, indicating a slower sedimentation rate. In contrast, the control group showed a significant increase in ΔBS in the bottom region over time (particle sedimentation), while the polysaccharide compound group's ΔBS remained almost stable. Furthermore, quantitative analysis using the Total Instability Index (TSI) showed that the CMC+GG compound group had the lowest TSI and the highest stability. Microrheological testing (…) Figure 15 or Figure 16 or Figure 17 The results show that the beverage with the compound polysaccharide system exhibits a higher viscosity index and elasticity index at low shear rates, while the flow index decreases, indicating that the system is more gel-like and viscous, which contributes to the long-term stability of suspended particles. Therefore, the beverage prepared by this invention possesses a uniform texture, high viscoelasticity, and excellent stability, and is less prone to sedimentation or stratification during pasteurization and subsequent storage.

[0126] In summary, this invention not only addresses the problem of peptide instability after sterilization in functional beverages by using CMC and GG as stabilizers to solve the utilization problem of enzymatically hydrolyzed peptides in functional beverages, but also employs stepwise enzymatic hydrolysis to prepare highly active ACE inhibitors. Furthermore, it utilizes bioinformatics methods and an online active peptide database to screen five new ACE-inhibiting peptides from enzymatically hydrolyzed egg yolks and elucidates the inhibition mechanism.

[0127] The specific embodiments described above do not constitute a limitation on the scope of protection of this disclosure. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of this disclosure.

Claims

1. An egg yolk polypeptide having ACE inhibitory activity, characterized in that: The amino acid sequence of the egg yolk polypeptide is any of the following: KFIPL, KFLPGY, KFLPTF, APTFGKL, KLPDMILY.

2. Use of the egg yolk polypeptide of claim 1 in the preparation of a functional beverage.

3. A functional beverage containing the egg yolk polypeptide with ACE inhibitory activity of claim 1.

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