Preparation method and application of novel leucine-rich egg white protein peptide

Abstract

The invention relates to the technical field of food science, and provides a preparation method and application of a novel leucine-rich egg white protein peptide.The preparation method comprises the steps that firstly, a protein structure is preliminarily destroyed through high-temperature preheating-acid hydrolysis two-dimensional pretreatment, and an internal hydrophobic structural domain is stretched; then, by applying an endonuclease and exonuclease synergistic enzymolysis strategy, the endonuclease accurately cuts a peptide bond in the protein, and the exonuclease orderly cuts from two ends of a peptide chain, so that target amino acid is efficiently exposed; in order to optimize the enzymolysis effect, a micro-jet high-pressure homogenization technology is integrated, an enzymolysis system passes through tiny slits at a high speed through ultrahigh pressure, a strong physical effect is generated, the contact of enzyme and a substrate is accelerated, the reaction rate is increased, and the particle size of an enzymolysis product is refined. Finally, the leucine is synthesized into a new peptide chain by utilizing Plastein reaction modification, so that the activity is improved. The process is safe, and the obesity relieving potential is huge.

Description

Technical Field

[0001] The present invention relates to the field of food science and technology, and specifically, to a preparation method and application of a novel leucine-rich egg white protein peptide. Background Art

[0002] Obesity has become a global public health problem and is closely related to various metabolic diseases (such as type 2 diabetes, cardiovascular diseases, and certain cancers). According to the data of the World Health Organization (WHO), the global obese population has increased significantly in the past few decades, especially in urban areas of developed and developing countries. Traditional obesity management methods, such as diet control, exercise intervention, and drug treatment, although effective to a certain extent, still have limitations, such as poor compliance and obvious side effects. Therefore, developing novel, safe, and effective obesity intervention strategies has become the focus of current research.

[0003] In recent years, bioactive peptides have received extensive attention due to their diverse physiological functions (such as antioxidant, anti-inflammatory, blood pressure-lowering, and metabolism-regulating effects, etc.). Compared with traditional drugs, bioactive peptides have the advantages of high safety, easy absorption, and strong targeting. In particular, functional peptides extracted from food proteins have become important raw materials for functional foods and nutritional supplements due to their natural sources and mature preparation processes. Research has shown that certain specific amino acid sequences (such as leucine-rich peptides) play a significant role in regulating energy metabolism, promoting fat decomposition, and inhibiting fat synthesis.

[0004] Egg white protein is a high-quality complete protein with the characteristics of high biological value, easy digestion and absorption, and balanced amino acid composition. It is rich in branched-chain amino acids (BCAAs) such as leucine, isoleucine, and valine, and these amino acids play important roles in regulating muscle synthesis, energy metabolism, and fat metabolism. In addition, egg white protein has a wide source, low cost, and its hydrolysis products have good solubility and stability, making it suitable for the development of functional foods.

[0005] Leucine, as an important branched-chain amino acid, has been proven to play a key role in regulating the mammalian target of rapamycin (mTOR) signaling pathway, thereby affecting protein synthesis, fat metabolism, and energy balance. Research has shown that leucine-rich peptides can slow down obesity through the following mechanisms: promoting fat oxidation: activating the AMP-activated protein kinase (AMPK) pathway and increasing fatty acid oxidation; inhibiting fat synthesis: downregulating the expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) to reduce fat accumulation; regulating appetite: by affecting the secretion of gastrointestinal hormones (such as GLP-1 and PYY), increasing satiety, and reducing energy intake.

[0006] In summary, although various preparation methods and functional activities of protein peptides have been reported in existing studies, there are still the following deficiencies in the preparation and application of leucine-rich peptides: 1) Insufficient purity and activity of peptides: Peptides prepared by existing methods often have a low leucine content and unstable functional activities; 2) Complex process and high cost: Some methods rely on expensive enzyme preparations or complex separation and purification steps, which limit their industrial applications; 3) Lack of application research for obesity. Existing studies mainly focus on the preparation of peptides and in vitro activity evaluation, lacking systematic in vivo functional verification and application development. Summary of the Invention

[0007] The content of the present invention is to provide a preparation method and application of a novel leucine-rich egg white protein peptide, which can successfully prepare a highly active egg white protein peptide with significant potential for reducing obesity by combining precise bioenzymatic technology with advanced physical modification means.

[0008] A novel preparation method of a leucine-rich egg white protein peptide according to the present invention comprises the following steps:

[0009] (1) Egg white separation: Select fresh eggs, clean the eggshells, break the eggs, separate the egg white and egg yolk, and filter with gauze to remove insoluble substances;

[0010] (2) Egg white dilution treatment: Add water in different proportions to the egg white in (1) to dilute the egg white, and the ratio of egg white to water is: 1:2 to 1:10;

[0011] (3) High-temperature preheating-acid treatment: Heat the diluted egg white protein solution at 80-95 °C for 10-30 min, maintain the pH at 2-5 and continuously stir during this process, and keep the rotation speed at 100-500 rpm;

[0012] (4) Enzyme treatment: Perform enzymatic hydrolysis on the solution in (3);

[0013] (5) Enzyme inactivation treatment: Perform enzyme inactivation treatment on the solution in (4), and centrifuge to take the supernatant;

[0014] (6) Particle refinement treatment: Use a shear-ultrasound-homogenization three-dimensional circulation technology to refine the protein particles in the solution in (5) to increase the surface area of the protein;

[0015] (7) Vacuum concentration treatment: Perform vacuum concentration treatment on the solution in (6), and the vacuum degree is 0.1-1 Pa;

[0016] (8) Plastein reaction: Perform a Plastein reaction on the protein solution in (7) with leucine;

[0017] (9) Enzyme inactivation: Heat the solution in (8) to inactivate the enzyme to terminate the reaction;

[0018] (10) Ultrafiltration process: Ultrafiltration is carried out using an ultrafiltration membrane with a molecular weight cut-off of 1 - 10 kDa;

[0019] (11) Dialysis: The solution in (10) is ultrafiltered to remove unreacted components;

[0020] (12) Freeze-drying: The solution in (11) is freeze-dried.

[0021] Preferably, in step (1), the stirring speed of the egg white is 50 - 300 r / min, and the stirring is carried out for 0.5 - 2 h;

[0022] In step (2), the egg white solution is diluted with water, and the ratio of egg white to water is 1:4 - 1:6.

[0023] Preferably, in step (3), the diluted egg white protein solution is heated at 90 - 95 °C for 20 min, and during this process, the pH is maintained at 3 - 4 and continuous stirring is carried out at a stirring speed of 400 rpm.

[0024] Preferably, in step (4), the enzymes are endoproteases and exoproteases. The endoproteases include papain, trypsin, proteinase K, bromelain, and flavorzyme, and the exoproteases include flavorzyme and leucine aminopeptidase;

[0025] The enzyme activity is 3000 - 8000 U / g pro, and the conditions for different enzymatic hydrolyses are as follows: the hydrolysis temperature of papain is 50 - 60 °C, and the pH is 6 - 7; the hydrolysis temperature of flavorzyme is 50 - 60 °C, and the pH is 6 - 7; the hydrolysis temperature of trypsin is 35 - 45 °C, and the pH is 7 - 9; the hydrolysis temperature of leucine aminopeptidase is 30 - 50 °C, and the pH is 7 - 9; the hydrolysis time is 30 - 60 min;

[0026] In step (5), inactivation of enzymes is carried out by treating at 80 - 95 °C for 10 - 25 min, and centrifugation is carried out at 5000 - 9000 rpm for 10 - 15 min.

[0027] Preferably, in step (6), the shear-ultrasound-homogenization three-dimensional circulation technology is a three-in-one of low-temperature high-speed shear - cell ultrasonic cavitation - high-pressure homogenization. Among them, the conditions for high-speed shear are: the rotation speed is 1000 - 5000 rpm, the cycle is carried out once every 1 - 5 min, and the cycle is carried out 3 - 5 times, and the temperature is maintained at 8 - 20 °C; then a low-temperature cell ultrasonic crusher is used to break the protein particles, the ultrasonic power is 10 - 60 W, the cycle is carried out once every 1 - 5 min, and the cycle is carried out 3 - 5 times; finally, homogenization is used to refine the particles, the pressure is 10 - 40 mPa, the cycle is carried out 3 times, and the temperature is controlled at 10 - 20 °C.

[0028] Preferably, in step (7), the egg white protease hydrolysate is concentrated under vacuum using a rotary evaporator, with a vacuum degree of 0.1 - 1 Pa, a temperature of 30 - 50 °C, a rotation speed of 30 - 90 rpm, and processed for 2 - 3 h.

[0029] Preferably, in step (8), the concentrated protein solution undergoes a Plastein reaction with leucine. The addition amount of leucine is 0.1 - 0.25%, the temperature is 30 - 50 °C, and the enzyme used in the Plastein reaction is selected from one of trypsin, chymotrypsin, papain, Bacillus subtilis, and thermolysin, and the reaction is carried out at a rotation speed of 200 rpm for 2 - 5 h.

[0030] Preferably, in step (9), after the Plastein reaction, the enzyme is inactivated at 85 - 95 °C to end the Plastein reaction process;

[0031] In step (10), the ultrafiltration membrane is 6 kDa or 8 kDa.

[0032] Preferably, in step (11), the ultrafiltrate is dialyzed using a dialysis bag with a molecular weight cut-off of 3500 Da to remove unreacted leucine, unreacted peptide fragments and other substances, and the dialysis time is 6 - 18 h;

[0033] In step (12), the conditions for freeze-drying are a temperature of -50 to -40 °C, a vacuum degree of 0.1 - 0.2 Pa, and a time of 32 - 38 h.

[0034] The present invention provides an application of a novel leucine-rich egg white protein peptide. The novel leucine-rich egg white protein peptide is obtained by using the above-mentioned preparation method of a novel leucine-rich egg white protein peptide and is used for the potential effect of alleviating obesity.

[0035] The present invention has the following beneficial effects:

[0036] 1) Innovation in the enzymatic hydrolysis process: The enzymatic hydrolysis process breaks through the tradition and introduces a number of advanced technologies. First, a two-dimensional pretreatment of "high-temperature preheating - acid hydrolysis" is adopted to preliminarily destroy the protein structure and unfold the internal hydrophobic domains. Then, a synergistic enzymatic hydrolysis strategy of endonuclease and exonuclease is used. The endonuclease precisely cuts the peptide bonds inside the protein, and the exonuclease cuts orderly from both ends of the peptide chain, efficiently exposing the target amino acids. To optimize the enzymatic hydrolysis effect, a microfluidic high-pressure homogenization technology is incorporated. Through ultra-high pressure, the enzymatic hydrolysis system passes through a tiny slit at a high speed, generating a strong physical effect, accelerating the contact between the enzyme and the substrate, increasing the reaction rate, and refining the particle size of the enzymatic hydrolysis products.

[0037] 2) Application of ultrasonic cavitation-shear effect: Refining the particle size of enzymolysate: The ultrasonic cavitation-shear effect is utilized to further refine the particle size of egg white protein enzymolysate, enhance its solubility and bioavailability, and provide a more uniform reaction matrix for the subsequent Plastein reaction.

[0038] 3) Modification of Plastein reaction: Amino acid synthesis into new peptide chains: Target amino acids (such as leucine) are synthesized into new peptide chains through the Plastein reaction to further increase the content of target amino acids, enhance the functional activity of peptides, especially their potential in alleviating obesity.

[0039] 4) Process safety: A safe and reliable preparation method, the entire process is safe and controllable, suitable for industrial production, and the prepared leucine-rich egg white protein peptides have high biological activity and functionality. Description of the drawings

[0040] Figure 1 It is a flowchart of a preparation method of a novel leucine-rich egg white protein peptide in Example 1.

[0041] Figure 2 It is a schematic diagram showing the influence of different enzyme treatments on the degree of hydrolysis of egg white protein in Example 1.

[0042] Figure 3 It is a schematic diagram of SDS-APGE of egg white protein treated with different enzymes in Example 1.

[0043] Figure 4 It is a schematic diagram showing the influence of different conditions on the particle size of egg white protein peptides in Example 1.

[0044] Figure 5 It is a schematic diagram showing the influence of egg white protein peptides treated with different methods on the body weight of HFD obese mice in Example 1.

[0045] Figure 6 It is a schematic diagram showing the influence of egg white protein peptides treated with different methods on the serum biochemistry of HFD obese mice in Example 1.

[0046] Figure 7 It is a schematic diagram showing the influence of egg white protein peptides treated with different methods on the adipose tissue pathology of HFD obese mice in Example 1; among them, A - F are the adipose tissue pathology sections of HFD obese mice in the control group, model group, egg white protein group, Example 1 group, comparative example 1, and comparative example 2 groups in sequence. Detailed implementation manners

[0047] To further understand the content of the present invention, the present invention will be described in detail in combination with the drawings and embodiments. It should be understood that the embodiments are only for explaining the present invention rather than limiting it.

[0048] Example 1

[0049] As Figure 1 shown, this embodiment provides a method for preparing a novel leucine-rich egg white protein peptide, which includes the following steps:

[0050] (1) Egg white separation: Select fresh eggs, clean the eggshells, break the eggs, separate the egg white and egg yolk, and filter with gauze to remove insoluble substances; the stirring speed of the egg white is 50-300 r / min, and stir for 0.5-2 h;

[0051] (2) Egg white dilution treatment: Add water in different proportions to the egg white in (1) to dilute the egg white, and the ratio of egg white to water is: 1:5;

[0052] (3) High-temperature preheating-acid treatment: Heat the diluted egg white protein solution at 95 °C for 20 min, and maintain the pH at 3 and continuously stir during this process, and keep the rotation speed at 400 rpm;

[0053] (4) Enzyme treatment: Perform enzymatic hydrolysis on the solution in (3); (The enzyme treatment is missing here)

[0054] In step (4), the enzymes are endoprotease and exoprotease. The endoproteases include papain, trypsin, proteinase K, bromelain, and flavorzyme. The exoproteases include flavorzyme and leucine aminopeptidase;

[0055] The enzyme activity is 3000-8000 U / g pro, and the conditions for different enzymatic hydrolyses are: the hydrolysis temperature of papain is 50-60 °C, and the pH is 6-7; the hydrolysis temperature of flavorzyme is 50-60 °C, and the pH is 6-7; the hydrolysis temperature of trypsin is 35-45 °C, and the pH is 7-9; the hydrolysis temperature of leucine aminopeptidase is 30-50 °C, and the pH is 7-9; the hydrolysis time is 30-60 min; this embodiment uses a combined enzymatic hydrolysis of flavorzyme and proteinase K;

[0056] (5) Enzyme inactivation treatment: Perform enzyme inactivation treatment on the solution in (4), and centrifuge to obtain the supernatant;

[0057] Perform enzyme inactivation treatment at 80-95 °C for 10-25 min, and centrifuge at 5000-9000 rpm for 10-15 min;

[0058] (6) Particle refinement treatment: Use the "shearing-ultrasonic-homogenization" three-dimensional circulation technology to refine the protein particles in the solution in (5) to increase the surface area of the protein;

[0059] The shear-ultrasound-homogenization three-dimensional circulation technology is a three-in-one of low-temperature high-speed shearing-cell ultrasonic cavitation-high-pressure homogenization. The high-speed shearing conditions are as follows: the rotation speed is 1000 - 5000 rpm, it circulates once every 1 - 5 minutes, circulates 3 - 5 times, and the temperature is maintained at 8 - 20 °C; then a low-temperature cell ultrasonic crusher is used to break the protein particles, the ultrasonic power is 10 - 60 W, it circulates once every 1 - 5 minutes, circulates 3 - 5 times; finally, the particles are refined by homogenization, the pressure is 10 - 40 mPa, it circulates 3 times, and the temperature is controlled at 10 - 20 °C;

[0060] (7) Vacuum concentration treatment: Perform vacuum concentration treatment on the solution in (6);

[0061] Use a rotary evaporator to perform vacuum concentration on the egg white protease hydrolysate, the vacuum degree is 0.1 - 1 Pa, the temperature is 30 - 50 °C, the rotation speed is 30 - 90 rpm, and it is treated for 2 - 3 h;

[0062] (8) Plastein reaction: Perform Plastein reaction on the protein solution in (7) and leucine;

[0063] The concentrated protein solution and leucine are subjected to Plastein reaction, the addition amount of leucine is 0.2%, the temperature is 40 °C, and the enzyme added in the Plastein reaction can be selected from one of trypsin, chymotrypsin, papain, Bacillus subtilis, and thermolysin. The enzyme conditions are as follows: the treatment conditions of trypsin are pH 7 - 8 and the temperature is 20 - 30 °C; the hydrolysis conditions of chymotrypsin are 7.0 - 9.0 and the temperature is 30 - 40 °C; the enzymatic hydrolysis conditions of papain are 5.0 - 7.0 and the temperature is 30 - 40 °C; the pH of Bacillus subtilis protease is 8.0 - 9.0 and the temperature is 40 - 50 °C, a mixed solution of the above components; in this example, papain is selected, the pH of the solution is adjusted to 7.5, and the reaction is carried out at a rotation speed of 400 rpm for 2 - 5 h;

[0064] (9) Enzyme inactivation: Heat the solution in (8) to inactivate the enzyme to terminate the reaction;

[0065] After the Plastein reaction, inactivate the enzyme at 85 - 95 °C to end the Plastein reaction process;

[0066] (10) Ultrafiltration process: Ultrafiltration is carried out with a 1 - 10 kDa ultrafiltration membrane; the ultrafiltration membrane is 6 kDa or 8 kDa;

[0067] (11) Dialysis: Ultrafilter the solution in (10) to remove unreacted components;

[0068] The ultrafiltrate is dialyzed with a 3500 Da dialysis bag to remove unreacted leucine, unreacted peptide fragments and other substances, and the dialysis time is 6 - 18 h;

[0069] (12) Freeze drying: The solution in (11) is freeze-dried;

[0070] The conditions for freeze drying are a temperature of -50 to -40 °C, a vacuum degree of 0.1 to 0.2 Pa, and a time of 32 - 38 h.

[0071] This example provides an application of a novel leucine-rich egg white protein peptide. The novel leucine-rich egg white protein peptide is obtained by using the preparation method of a novel leucine-rich egg white protein peptide described above and is used for the potential effect of alleviating obesity.

[0072] Example 2

[0073] The difference between this example and Example 1 is that in step (4), a composite enzyme hydrolysis using flavor protease and bromelain is used, and other operations are the same as in Example 1.

[0074] Example 3

[0075] The difference between this example and Example 1 is that in step (4), a stepwise hydrolysis using proteinase K and leucine aminopeptidase is used, and other operations are the same as in Example 1.

[0076] Example 4

[0077] The difference between this example and Example 1 is that in step (4), a stepwise hydrolysis using bromelain and leucine aminopeptidase is used, and other operations are the same as in Example 1.

[0078] Example 5

[0079] The difference between this example and Example 1 is that in step (4), a simultaneous hydrolysis using bromelain and leucine aminopeptidase is used, and other operations are the same as in Example 1.

[0080] Example 6

[0081] The difference between this example and Example 1 is that in step (8), the enzyme added in the Plastein reaction is treated with trypsin, and other operations are the same as in Example 1.

[0082] Example 7

[0083] The difference between this example and Example 1 is that in step (8), the enzyme added in the Plastein reaction is treated with chymotrypsin, and other operations are the same as in Example 1.

[0084] Example 8

[0085] This example conducts a test analysis, and the test method is as follows:

[0086] 1. Determination of degree of hydrolysis

[0087] The hydrolysis degree of the protein solution was determined by the o-phthalaldehyde method (OPA method). The hydrolysis degree was calculated according to Equation (1):

[0088]

[0089] where: N is the concentration of amino nitrogen in the hydrolyzate, mol / L; N0 is the concentration of amino nitrogen in the protein solution, mol / L; ρ is the sample mass concentration (5 mg / mL); h tot is the total number of peptide bonds in the protein, mmol / g, and in this example, h tot is 8.0.

[0090] 2. Determination of SDS-PAGE

[0091] The reduced SDS-PAGE of the egg white protein hydrolysate sample was analyzed. 15 μL of each 2 mg / mL sample was added to the gel. A 15% separating gel and a 5% stacking gel were selected for electrophoresis. The initial voltage of electrophoresis was 120 V, and when the sample entered the separating gel, the voltage was increased to 160 V.

[0092] 3. Determination of particle size

[0093] The sample solution with a concentration of 1 mg / mL was prepared by fully mixing the sample with phosphate buffer solution. 1 mL was taken and placed in a test dish, and the particle size of the sample was measured using a laser particle size analyzer.

[0094] 4. Determination of leucine content

[0095] A certain amount of egg white protein powder was weighed and dissolved in deionized water to 10 mg / mL. 2 mL was taken, 3 mL of ninhydrin solution and 0.1 mL of 0.3% ascorbic acid solution were added. After mixing, it was boiled in boiling water for 15 min, then cooled in cold water and shaken well. The sample extract was taken, and 5 mL was centrifuged at 8000 r / min for 10 min. The absorbance value of the supernatant was measured at a wavelength of 580 nm. Using leucine as a reference substance, a standard curve was plotted with absorbance as the ordinate and concentration as the abscissa, and the content of the sample could be read from the standard curve.

[0096] 5. Lipase inhibitory activity

[0097] Pre-incubate at 37 °C for 20 minutes, consisting of 20 μL of samples (peptide at concentrations of 1, 0.5, 0.25, 0.2, 0.1, 0.05, 0.025, 0.00125, and 0.00625 mg / mL or orlistat at 10, 5, 4, 3, 2, 1, and 0.5 μM) and 50 μL of lipase inhibitory activity (1 mg / mL) in 100 mM PBS. Subsequently, add 50 μL of p-nitrophenyl butyrate substrate (5 mM) to initiate the incubation reaction for 10 min. Measure the absorbance at 405 nm to determine the inhibition rate.

[0098] 6. Lipocholesterolase inhibitory activity

[0099] Incubate peptide samples at various concentrations (each 0.5, 0.25, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 mg / mL, 20 μL) with lipocholesterolase (1 U / mL in 100 mM PBS) at 37 °C for 20 min. Subsequently, add 100 μL of 100 mM PBS buffer containing taurocholic acid (5.16 mM) and p-nitrophenyl butyrate (4 mM) to initiate the reaction. Measure the absorbance at 405 nm in a 96-well plate after 10 minutes using a multi-functional microplate reader (Thermo Scientific, New York, USA) to determine the inhibition rate. Use orlistat at the designed concentrations (10, 5, 4, 3, 2, 1, 0.5 μM) as a positive control.

[0100] 7. Animal experiment design

[0101] C57BL / 6J male mice (3 - 4 weeks old) were acclimated for 7 days in a standard light / dark cycle, with free access to food and water, and then divided into 6 groups: normal diet group, model group, Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group; among them, the model group, Example 1 group, Example 2 group, Comparative Example 1 group, and Comparative Example 2 group were given a 60% high-fat diet feed, while the normal diet group was given a normal maintenance feed. Starting from the 7th week, the above-mentioned model group, Example 1 group, Example 2 group, Comparative Example 1 group, and Comparative Example 2 group were respectively given the egg white protein hydrolysate prepared by the corresponding method at 600 mg / mL, while the normal diet group was given an equal amount of normal saline. Record the body weight of all mice weekly. At the end of the 13-week experiment, all mice were bled by orbital puncture after fasting for 12 hours, and each mouse was sacrificed by cervical dislocation. The mouse blood taken was allowed to stand at 4 °C for 12 h to separate the serum, centrifuged at 10000 rpm for 10 min, and the upper serum was collected. Subsequently, the epididymal adipose tissue was separated and collected, and fixed in 4% formaldehyde solution.

[0102] 8. Serum biochemical analysis

[0103] The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in serum were measured using the kits from Nanjing Jiancheng Reagent Co., Ltd.

[0104] 9. Histopathological analysis of adipose tissue

[0105] Fresh epididymal adipose tissue was fixed in 4% PFA (polytetrafluoroethylene soluble) for 24 hours. Alcohol with increasing concentration was used as a dehydrating agent to gradually dehydrate the tissue. Then the tissue blocks were embedded in paraffin, and xylene was used instead of ethanol as a clearing agent. Then the paraffin blocks were tightly mounted on a microtome and sectioned at 5 μm. The tissue samples were stained with hematoxylin-eosin (HE). First, the sections were immersed in distilled water, and then stained with hematoxylin for 3 min. After staining, the sections were treated with acid and ammonia, and then rinsed with tap water. Dehydration was carried out in two stages, 10 min for each stage, using 70% and 90% ethanol. Then the sections were immersed in eosin solution for 3 min. Finally, the sections were dehydrated in absolute ethanol containing xylene and sealed for observation.

[0106] The following comparative examples were used for comparison in this example:

[0107] Comparative Example 1

[0108] (1) Egg white separation: Select fresh eggs, clean the eggshells, break the eggs, separate the egg white and egg yolk, maintain a certain speed for a certain time, and filter with gauze to remove insoluble substances;

[0109] (2) Egg white dilution treatment: Different proportions of water were added to the egg white in (1) to dilute the egg white, and the ratio of egg white to water was 1:5.

[0110] (3) High-temperature preheating-acid treatment: The diluted egg white protein solution was heated at 95 °C for 20 min, and during this process, the pH was maintained at 4 and continuously stirred, and the rotation speed was maintained at 400 rpm;

[0111] (4) Enzyme treatment: The solution in (3) was enzymatically digested;

[0112] (5) Enzyme inactivation treatment: The solution in (4) was inactivated by enzymes, and the supernatant was taken by centrifugation;

[0113] (6) Particle refinement treatment: The solution in (5) was refined using the "shearing-ultrasonic-homogenization" three-dimensional circulation technology;

[0114] (7) Freeze-drying: The solution in (6) was freeze-dried;

[0115] Comparative Example 2

[0116] (1) Egg white separation: Select fresh eggs. After cleaning the eggshells, break the eggs and separate the egg whites and yolks. Maintain a certain speed for a certain period of time, and filter with gauze to remove insoluble substances;

[0117] (2) Egg white dilution treatment: Add water in different proportions to the egg white in (1) to dilute the egg white. The ratio of egg white to water is 1:5.

[0118] (3) High-temperature preheating - acid treatment: Heat the diluted egg white protein solution at 95 °C for 20 min, and maintain the pH at 3 and stir continuously during this process, and keep the rotation speed at 400 rpm;

[0119] (4) Particle refinement treatment: Use the "shearing - ultrasonic - homogenization" three-dimensional circulation technology to refine the protein particles in the solution in (3);

[0120] (5) Vacuum concentration treatment: Conduct vacuum concentration treatment on the solution in (4), and the vacuum degree is 0.1 - 1 Pa.

[0121] (6) Plastein reaction: Conduct Plastein reaction on the protein solution in (5) with leucine;

[0122] (7) Enzyme inactivation: Heat the solution in (6) to inactivate the enzyme to terminate the reaction;

[0123] (8) Ultrafiltration process: Conduct ultrafiltration with a 6 kDa or 8 kDa ultrafiltration membrane;

[0124] (9) Dialysis: Ultrafilter the solution in (8) to remove unreacted components;

[0125] (10) Freeze-drying: Conduct freeze-drying on the solution in (9);

[0126] Comparative Example 3

[0127] (1) Egg white separation: Select fresh eggs. After cleaning the eggshells, break the eggs and separate the egg whites and yolks. Maintain a certain speed for a certain period of time, and filter with gauze to remove insoluble substances;

[0128] (2) Egg white dilution treatment: Add water in different proportions to the egg white in (1) to dilute the egg white. The ratio of egg white to water is: 1:5.

[0129] (3) High-temperature preheating - acid treatment: Heat the diluted egg white protein solution at 95 °C for 20 min, and maintain the pH at 3 and stir continuously during this process, and keep the rotation speed at 400 rpm;

[0130] (4) Enzyme treatment: Conduct enzymatic hydrolysis treatment on the solution in (3);

[0131] (5) Enzyme inactivation treatment: Conduct enzyme inactivation treatment on the solution in (4), and centrifuge to take the supernatant;

[0132] (6) Particle refinement treatment: The solution in (5) is subjected to a "shearing - ultrasonic - homogenization" three - dimensional circulation technique to refine protein particles;

[0133] (7) Vacuum concentration treatment: The solution in (6) is subjected to vacuum concentration treatment, and the vacuum degree is 0.1 - 1 Pa.

[0134] (8) Plastein reaction: The protein solution in (7) undergoes a Plastein reaction without adding leucine;

[0135] (9) Enzyme inactivation: The solution in (8) is heated to inactivate the enzyme to terminate the reaction;

[0136] (10) Ultrafiltration process: Ultrafiltration is carried out using a 6 kDa or 8 kDa ultrafiltration membrane;

[0137] (11) Dialysis: The solution in (10) is ultrafiltered to remove unreacted components;

[0138] (12) Freeze - drying: The solution in (11) is freeze - dried;

[0139] Comparative Example 4

[0140] Omit the particle refinement treatment in step (6) of Example 1, and the others are the same as Example 1.

[0141] Comparative Example 5

[0142] Omit the vacuum concentration treatment in (7) and the Plastein reaction in (8) of Example 1, and the others are the same as Example 1.

[0143] Comparative Example 6

[0144] Omit the enzyme treatment in (4), the enzyme inactivation treatment in (5) and the particle refinement treatment in (6) of Example 1, and the others are the same as Example 1.

[0145] Analysis of the influence of different enzyme treatments on the degree of hydrolysis of egg white protein

[0146] The degree of protein hydrolysis not only reflects the degradation degree of proteins during enzymatic hydrolysis but also directly affects the functional properties (such as solubility, emulsifying property, and biological activity) and application value of the products. Enzymes degrade macromolecular proteins into small peptide segments or amino acids by specifically or non-specifically cleaving peptide bonds, and their cleavage sites and efficiencies are affected by the types of enzymes, substrate structures, and reaction conditions (such as pH and temperature). For example, endopeptidases act on the interior of peptide chains, and exopeptidases gradually cleave from the ends of peptide chains. The synergistic action of the two can significantly improve the hydrolysis efficiency. Understanding the effects of different enzyme treatments on the degree of hydrolysis of egg white proteins helps optimize the enzymatic hydrolysis process, obtain specific functional peptide segments, and provide theoretical basis and technical support for the development of high-value-added functional foods (such as anti-obesity peptides and antioxidant peptides), promoting the efficient utilization of protein resources and the innovative development of functional foods. The effects of different enzyme treatments on the degree of hydrolysis of egg white proteins are as Figure 2 shown. As Figure 2 can be seen, after treating egg white proteins with different enzymes, their hydrolysates are significant. This may be due to the different cleavage sites of different enzymes and the differences in their affinity for specific proteins in egg white proteins. As can be seen from the figure, the hydrolysis degrees of bromelain, proteinase K, papain, and flavor protease on egg white proteins are relatively high, all exceeding 15%, and the hydrolysis degree of proteinase K is the highest. In addition, with the extension of the enzymatic hydrolysis time, the degree of hydrolysis of egg white proteins all increases. The differences in protein enzymatic hydrolysis may be affected by factors such as enzyme type, enzymatic hydrolysis time, enzyme type, temperature, and pH. Among them, enzyme type and enzymatic hydrolysis time, as important factors affecting the degree of protein hydrolysis, are crucial for screening different proteases and optimizing the enzymatic hydrolysis conditions. More importantly, the enzymatic hydrolysis conditions are also the key factors limiting the physiological activity of enzymatic hydrolysis products. Hydrolysis may release bioactive peptide segments, such as antioxidant and antihypertensive peptides. Hydrolysis exposes the active sequences that were originally hidden in the protein structure.

[0147] SDS-APGE of egg white proteins treated with different enzymes

[0148] Through SDS-PAGE, not only can the band distribution and approximate range of proteins or protein peptides be observed more intuitively, but also the changes in the bands of proteins or protein peptides after treatment can be observed. The SDS-APGE of egg white proteins treated with different enzymes is as Figure 3 shown. As Figure 3It can be seen that there are significant differences in the band migration and corresponding molecular weights of egg white proteins treated with different enzymes. Untreated egg white protein has 5 bands, namely ovomucoid, ovotransferrin, ovalbumin, ovomucin, and lysozyme in sequence. When hydrolyzed with different enzymes, the relevant bands of the original egg white protein changed significantly. Among them, the most significant changes were that the bands of ovomucoid, ovotransferrin, and ovalbumin were diffused or even disappeared and migrated below 52 kDa. Among the hydrolysis products of all enzymes, bromelain, proteinase K, and papain treatments had smaller molecular weights. This result is also consistent with the above results of the degree of hydrolysis. Therefore, bromelain, proteinase K, and papain were selected for subsequent preparation of egg white protein hydrolysates in this example.

[0149] Effect of different conditions on the particle size of egg white protein peptides

[0150] Particle size has a significant impact on the properties and functions of proteins and protein peptides. A smaller particle size generally means that protein molecules are more dispersed and have a higher surface hydrophobicity, which helps to improve the solubility and stability of proteins, and vice versa. In addition, changes in particle size also affect the transport and interaction of proteins in vivo, such as intracellular localization and function. Particle size determination is of great significance for the research and application of proteins and protein peptides. Therefore, the particle size differences of egg white protein peptides prepared under different conditions are as Figure 4 shown. As Figure 4 can be seen, there are significant differences in the particle sizes of egg white protein peptides prepared under different conditions. Compared with Comparative Example 2, the particle sizes of the remaining groups are smaller and more evenly dispersed. More importantly, after the Plastein reaction of leucine with the enzymatically hydrolyzed egg white protein peptides, the particle size of the egg white protein peptides increased slightly, but the change was not significant. This may be because the exogenously added leucine covalently bonded with the egg white protein peptides, thus increasing the molecular weight of the original egg white protein hydrolysate. During this process, due to the existence of certain non-covalent interactions such as hydrogen bonds and electrostatic interactions, it may improve its solubility in water and increase its dispersibility.

[0151] Leucine content of egg white protein peptides treated differently

[0152] Leucine is an essential amino acid and one of the branched-chain amino acids (BCAAs), which is widely present in foods such as animal proteins, dairy products, and legumes. Leucine has many important functions in the body, including promoting muscle synthesis, reducing muscle protein breakdown, regulating energy metabolism, improving blood glucose control, and modulating the gut microbiota. Determining the leucine content in foods is of great significance for evaluating protein quality, designing food formulations, ensuring the efficacy and safety of nutritional supplements, and supporting biomedical research. At the same time, it helps to promote healthy eating and prevent chronic diseases. In summary, as an important essential amino acid, leucine has multiple important significances in the fields of human health and food science. Accurately determining the leucine content in foods is of great significance for evaluating food nutritional value, guiding reasonable diet, and supporting related scientific research. The leucine content of egg white protein peptides is shown in Table 1. As can be seen from the table, there are significant differences in the leucine content of egg white protein peptides under different treatments. Compared with Comparative Example 1, the leucine content of each Example is higher, which may be due to the Plastein reaction between the egg white protein hydrolysate and exogenous leucine, and the covalent reaction between leucine and egg white protein peptides, resulting in an increase in the leucine content. In addition, compared with Comparative Example 3, the leucine content of the egg white protein peptides prepared in Example 1 is also more. This also indicates that exogenous addition of leucine through the Plastein reaction can better enrich the leucine content in protein peptides with concentrated egg white protein hydrolysates.

[0153] Table 1 Leucine content of egg white protein peptides under different treatments

[0154]

[0155]

[0156] Lipase inhibitory activity of egg white protein peptides under different treatments

[0157] Lipase is a key enzyme that catalyzes the hydrolysis of fats. The inhibition of its activity can effectively reduce the absorption and accumulation of fats, thus having potential application value in controlling obesity and related metabolic diseases. By measuring the lipase inhibitory activity, potential natural or synthetic inhibitors can be screened and evaluated, providing a scientific basis for the development of weight-loss drugs and functional foods. Protein peptides have the characteristics of wide sources, high safety, and good biocompatibility, making them ideal candidates for lipase inhibitors. By measuring the lipase inhibitory activity of protein peptides, the type and intensity of their inhibition of lipase can be determined, thus providing guidance for optimizing the structure and function of protein peptides. The lipase inhibitory activities of egg white protein peptides treated differently are shown in Table 2. As can be seen from the table, there are significant differences in the lipase inhibitory activities of egg white protein peptides treated differently. Compared with the egg white protein group, the inhibitory activity of the treated egg white protein peptides against lipase is significantly enhanced, and the inhibitory effect of Example 1 is the best, which may be due to the enrichment of more leucine in Example 1, thus enhancing its ability to inhibit lipase activity.

[0158] Table 2 Lipase Inhibitory Activities of Egg White Protein Peptides Treated Differently

[0159]

[0160]

[0161] Cholesterol Esterase Inhibitory Activities of Egg White Protein Peptides Treated Differently

[0162] Cholesterol esterase plays a key role in lipid metabolism, and the regulation of its activity is crucial for maintaining the balance of cholesterol and triglyceride levels in the body. The determination of cholesterol esterase inhibitory activity can help evaluate the impact of potential inhibitors on cholesterol metabolism, providing a scientific basis for the development of drugs and functional foods that regulate blood lipid levels. For example, inhibiting the activity of cholesterol esterase can reduce the absorption and synthesis of cholesterol, thus lowering the level of low-density lipoprotein cholesterol (LDL-C) in the blood and preventing atherosclerotic cardiovascular disease (ASCVD). Therefore, it is of great significance to measure the inhibitory activity of egg white protein peptides against cholesterol esterase. The cholesterol esterase inhibitory activities of egg white protein peptides treated differently are shown in Table 3. As can be seen from the table, there are significant differences in the inhibitory activities of egg white protein peptides treated differently against cholesterol esterase. Compared with the egg white protein group, the inhibitory activity of the treated egg white protein peptides against cholesterol esterase is significantly enhanced, and the inhibitory effect of Example 1 is the best, which may be due to the enrichment of more leucine in Example 1, thus enhancing its ability to inhibit cholesterol esterase activity. In summary, based on the results of the lipase inhibitory activity and cholesterol esterase inhibitory activity of egg white protein, egg white protein, Example 1, Comparative Example 1, and Comparative Example 2 were selected for subsequent animal experiments.

[0163] Table 3 Lipocholesterol esterase inhibitory activity of egg white protein peptides with different treatments

[0164]

[0165]

[0166] Effect of egg white protein peptides with different treatments on the body weight of HFD obese mice

[0167] Obesity is a complex metabolic disease, and its occurrence is not only related to the imbalance of energy metabolism, but also accompanied by extensive molecular-level changes, such as inflammation and lipid metabolism disorders. By protein administration, the body weight of obese mice can be regulated, and then their metabolic disorder state can be improved. For example, certain proteins or protein peptides can reduce the body weight of obese mice through mechanisms such as regulating appetite, promoting fat decomposition, and improving insulin sensitivity. The change in body weight is one of the direct indicators for obesity treatment, and the regulatory effect of proteins / protein peptides on body weight may involve multiple levels. Therefore, it is of great significance to explore the effect of protein intake on the body weight of HFD obese mice. The effect of egg white protein peptides with different treatments on the body weight of HFD obese mice after 13 weeks of feeding is as Figure 5 shown. As can be seen from Figure 5 , compared with the control group, the body weights of the remaining groups all increased significantly, especially the body weight of the model group increased most significantly. Compared with the model group, the body weights of HFD obese mice decreased significantly after ingesting egg white protein. This indicates that egg white protein peptides have the effect of slowing down obesity. The egg white protein peptides prepared in Example 1 had the most significant effect on reducing the body weight of HFD obese mice. This may be because exogenous leucine was added to carry out the Plastein reaction with the original egg white protein peptides, so that more leucine was covalently bonded in the protein peptides, thereby enhancing their effect of alleviating obesity.

[0168] Effect of egg white protein peptides with different treatments on the serum biochemistry of HFD obese mice

[0169] It is of great significance to study the effect of protein administration on the serum biochemical indexes of high-fat diet (HFD)-induced obese mice. Obesity is not only manifested as an increase in body weight, but also accompanied by a series of metabolic disorders, such as dyslipidemia, elevated blood glucose, and inflammatory responses. Exploring the significance of serum biochemical indexes for the alleviation of obesity by proteins / protein peptides helps to comprehensively evaluate the potential value of proteins / protein peptides in obesity treatment. The change in serum biochemical indexes can reflect the regulatory effect of proteins / protein peptides on the metabolic state of obese mice. The effect of egg white protein peptides with different treatments on the serum biochemistry of HFD obese mice is as Figure 6 shown. As can be seen from Figure 6It can be seen that compared with the control group, the serum lipid levels (TG, TC, HDL-c, LDL-c) of the remaining groups were significantly increased. However, compared with the model group, the serum lipid levels of the experimental group given egg white protein peptides were significantly decreased. This indicates that the intake of egg white protein reversed the serum lipid levels of HFD obese mice. The effect of Example 1 in reversing the serum lipid levels of HFD obese mice was the most significant, which may be due to the combined action of endo-exopeptidase hydrolysis and Plastein reaction, not only making leucine in egg white protein more effectively exposed, but also increasing the enrichment amount of leucine in egg white protein. Relevant literature shows that leucine has great potential in regulating obesity.

[0170] Effects of Egg White Protein Peptides with Different Treatments on Adipose Tissue Pathology of HFD Obese Mice

[0171] Adipose tissue is not only the main site of energy storage, but also participates in metabolic regulation by secreting various adipokines (such as leptin, adiponectin, etc.). Protein supplementation can regulate the metabolic function of adipose tissue, improve the inflammatory state of adipose tissue, and thus affect the whole-body metabolic health of obese mice. The structural and functional changes of adipose tissue are one of the key factors in the occurrence and development of obesity. Therefore, studying the effects of protein supplementation on adipose tissue in HFD obese mice and the mechanism of action of adipose tissue on the alleviation of obesity by proteins / protein peptides not only helps to reveal the pathophysiology of obesity, but also provides an important theoretical basis for the development of protein / protein peptide-based obesity treatment strategies. The effects of egg white protein peptides with different treatments on adipose tissue pathology of HFD obese mice are as Figure 7 shown. As Figure 7 can be seen, compared with the control group, the cells in the adipose tissue of the remaining groups were larger and showed a disordered distribution; while compared with the model group, the adipose cells of HFD obese mice given egg white protein peptides were significantly smaller and arranged more evenly. This result also indicates that the intake of egg white protein peptides helps to reverse the trend of adipocyte hypertrophy in HFD obese mice. The effect of Example 1 in alleviating adipocyte hypertrophy was the most significant. This may be because the Plastein reaction occurred between the egg white protein peptides and exogenous leucine, enriching more leucine on the original egg white protein peptides, thus endowing them with stronger potential to alleviate obesity.

[0172] The present invention and its embodiments are schematically described above. The description is not restrictive, and only one of the embodiments of the present invention is shown in the drawings. The actual structure is not limited thereto. Therefore, if those of ordinary skill in the art are inspired by it and design similar structural modes and embodiments to this technical solution without creative efforts without departing from the purpose of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. A preparation method of a novel leucine-rich egg white protein peptide, characterized in that, It includes the following steps: (1) Egg white separation: Select fresh eggs, clean the eggshells, break the eggs, separate the egg whites and yolks, and filter with gauze to remove insoluble substances; (2) Dilution treatment of egg white: Add water in different proportions to the egg white in (1) to dilute the egg white. The ratio of egg white to water is 1:2 - 1:10; (3) High-temperature preheating - acid treatment: Heat the diluted egg white protein solution at 80 - 95°C for 10 - 30 min, maintain the pH at 2 - 5 and stir continuously during this process, and keep the rotation speed at 100 - 500 rpm; (4) Enzyme treatment: Enzymatically hydrolyze the solution in (3); (5) Enzyme inactivation treatment: Perform enzyme inactivation treatment on the solution in (4), and centrifuge to obtain the supernatant; (6) Particle refinement treatment: Use a shear - ultrasonic - homogenization three - dimensional circulation technology to refine the protein particles in the solution in (5) to increase the surface area of the protein; (7) Vacuum concentration treatment: Perform vacuum concentration treatment on the solution in (6), and the vacuum degree is 0.1 - 1 Pa; (8) Plastein reaction: Perform a Plastein reaction on the protein solution in (7) with leucine; (9) Enzyme inactivation: Heat the solution in (8) to inactivate the enzyme to terminate the reaction; (10) Ultrafiltration process: Ultrafilter with a 1 - 10 kDa ultrafiltration membrane; (11) Dialysis: Ultrafilter the solution in (10) to remove unreacted components; (12) Freeze - drying: Perform freeze - drying on the solution in (11).

2. The preparation method of a novel leucine-rich egg white protein peptide according to claim 1, characterized in that, In step (1), the stirring speed of the egg white is 50 - 300 r / min, and stir for 0.5 - 2 h; In step (2), dilute the egg white solution with water, and the ratio of egg white to water is 1:4 - 1:

6.

3. The preparation method of a novel leucine-rich egg white protein peptide according to claim 1, characterized in that, In step (3), heat the diluted egg white protein solution at 90 - 95°C for 20 min, maintain the pH at 3 - 4 and stir continuously during this process, and the stirring speed is 400 rpm.

4. The preparation method of a novel leucine-rich egg white protein peptide according to claim 1, characterized in that, In step (4), the enzymes are endoprotease and exoprotease. The endoproteases include papain, trypsin, proteinase K, bromelain, and flavorzyme. The exoproteases include flavorzyme and leucine aminopeptidase; The enzyme activity is 3000 - 8000 U / g pro. The conditions for different enzymatic hydrolyses are: the hydrolysis temperature of papain is 50 - 60°C, and the pH is 6 - 7; the hydrolysis temperature of flavorzyme is 50 - 60°C, and the pH is 6 - 7; the hydrolysis temperature of trypsin is 35 - 45°C, and the pH is 7 - 9; the hydrolysis temperature of leucine aminopeptidase is 30 - 50°C, and the pH is 7 - 9; the hydrolysis time is 30 - 60 min; In step (5), perform enzyme inactivation treatment at 80 - 95°C for 10 - 25 min, and centrifuge at 5000 - 9000 rpm for 10 - 15 min.

5. The preparation method of a novel leucine-rich egg white protein peptide according to claim 1, characterized in that, In step (6), the shear-ultrasound-homogenization three-dimensional circulation technology is a three-in-one of low-temperature high-speed shearing-cellular ultrasound cavitation-high-pressure homogenization. Among them, the high-speed shearing conditions are as follows: the rotation speed is 1000-5000 rpm, it circulates once every 1-5 minutes, circulates 3-5 times, and the temperature is maintained at 8-20 °C; then a low-temperature cell ultrasonic crusher is used to crush the protein particles, the ultrasonic power is 10-60 W, it circulates once every 1-5 minutes, and circulates 3-5 times; finally, the particles are refined by homogenization, the pressure is 10-40 mPa, it circulates 3 times, and the temperature is controlled at 10-20 °C.

6. The preparation method of a novel leucine-rich egg white protein peptide according to claim 1, characterized in that, In step (7), a rotary evaporator is used to vacuum-concentrate the egg white protease hydrolysate. The vacuum degree is 0.1-1 Pa, the temperature is 30-50 °C, the rotation speed is 30-90 rpm, and it is processed for 2-3 h.

7. The preparation method of a novel leucine-rich egg white protein peptide according to claim 1, characterized in that In step (8), the concentrated protein solution and leucine undergo a Plastein reaction. The addition amount of leucine is 0.1-0.25%, the temperature is 30-50 °C, and the enzyme added in the Plastein reaction is selected from one of trypsin, chymotrypsin, papain, Bacillus subtilis, and thermolysin, and the reaction is carried out at a rotation speed of 200 rpm for 2-5 h.

8. The preparation method of a novel leucine-rich egg white protein peptide according to claim 1, characterized in that, In step (9), after the Plastein reaction, the enzyme is inactivated at 85-95 °C to end the Plastein reaction process; In step (10), the ultrafiltration membrane is 6 kDa or 8 kDa.

9. The preparation method of a novel leucine-rich egg white protein peptide according to claim 1, characterized in that, In step (11), the ultrafiltrate is dialyzed using a dialysis bag with a molecular weight cut-off of 3500 Da to remove substances such as unreacted leucine and unreacted peptide fragments. The dialysis time is 6-18 h; In step (12), the conditions for freeze-drying are a temperature of -50 to -40 °C, a vacuum degree of 0.1-0.2 Pa, and a time of 32-38 h.

10. Application of a novel leucine-rich egg white protein peptide, characterized in that, The novel leucine-rich egg white protein peptide is obtained by using the preparation method of a novel leucine-rich egg white protein peptide described in any one of claims 1-9, and is used for the potential effect of alleviating obesity.

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