Preparation method and application of novel egg white protein muscle building powder

Abstract

The invention relates to a preparation method and application of novel egg white protein muscle building powder, and provides high-quality nutrition support for improving muscle quality. The method takes fresh egg white as a raw material; a finished product is prepared through the steps of egg white separation after eggshell disinfection, ultrasonic-shearing synergetic flocculation removal, yeast beta glucan-polyphenol composite microsphere live protection system addition, acid-dynamic high-pressure microjet low-temperature pretreatment, compound protease enzymolysis, ultrafiltration impurity removal, gradient pH membrane separation and purification, low-temperature vacuum spray drying and the like. The composite microspheres are adopted to replace chemical protecting groups, and the whole-course low-temperature processing technology is combined, so that the activity of branched chain amino acid is effectively maintained. The protein powder can prolong the exercise exhaustion time of mice with muscle injury, improve the muscle repair efficiency, and realize the dual functions of muscle-building nutrition and oxidation resistance. The product adapts to various food systems, and can be widely applied to the fields of sports nutrition and food for special medical purposes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine and special medical food, and particularly relates to a preparation method and application of a novel egg white protein muscle building powder. BACKGROUND

[0002] In muscle growth of the elderly and muscle repair of the fitness population, efficient activation of muscle synthesis (MPS) is the core goal of muscle building, and essential amino acids provided by protein supplements are the key material basis for muscle repair and growth. Among them, branched chain amino acids (BCAA, containing leucine Leu, isoleucine Ile and valine Val) are the core signal molecules for regulating muscle synthesis, and their structural integrity and activity directly determine the muscle building efficiency - especially leucine, which can specifically activate the mTOR signaling pathway and is the key switch to start muscle protein synthesis. However, the current commercially available protein supplements generally have low bioavailability, incomplete amino acid spectrum and other problems, and the core bottleneck is that the retention rate and activity of BCAA are insufficient, which is difficult to meet the high-efficiency muscle building demand of the fitness population and special demand groups.

[0003] Due to the characteristics of high protein, low fat and complete amino acid spectrum, egg white protein becomes an ideal substrate for developing BCAA-rich products, but its industrial application is limited by the inherent defects of traditional processing technology. Traditional egg white protein production often uses high-temperature hot air drying and long-time alkaline hydrolysis process, which lacks a targeted BCAA protection mechanism and easily causes double irreversible damage to BCAA. On the one hand, high temperature or alkaline environment can cause the side chain methyl of Leu, Ile and Val to be oxidized and broken, generating inactive derivatives such as hydroxy acid and ketone acid, which lose the structural basis for recognition by the mTOR pathway. On the other hand, high temperature can induce the conformational reconstruction of BCAA peptide bond, from the exposed hydrophilic state to the embedded hydrophobic state, which cannot effectively bind to the hydrophobic active site of mTORC1, significantly reducing the downstream signal phosphorylation efficiency and weakening the muscle synthesis signal transduction. At the same time, traditional processes rely on chemical protectants to improve BCAA retention, which has residual risks and cannot consider product functionality, further limiting product application scenarios. Therefore, developing an egg white protein processing technology with the characteristics of "efficiently enriching BCAA, fully protecting activity, and being green and residue-free" has become a key problem that needs to be broken through in the industry. SUMMARY

[0004] The present application aims to provide a preparation method and application of a novel egg white protein muscle building powder, which solves the problems of low bioavailability of existing protein supplements, insufficient BCAA activity and the core pain points of traditional egg white protein processing technology "difficult to enrich, easy to lose activity and residual". The present application aims to provide efficient and safe muscle building nutritional support for the fitness population and special medical use demand groups. Based on the advantages of high protein and low fat of egg white protein and the high content of leucine, efficient enrichment and active complete retention of BCAA are achieved.

[0005] To solve the above technical problems, the technical scheme adopted by the present application is: A preparation method of a novel egg white protein muscle growth powder, comprising the following steps: (1) Egg white separation: select fresh eggs, sterilize the eggshells with ethanol, break the shells, and separate the egg white and yolk; stir the egg white to make the egg white system uniform; (2) Ultrasonic-shearing treatment of egg white to obtain deflocculated egg white; (3) Add deionized water to dilute the deflocculated egg white, add yeast β-glucan composite microspheres, and stir at constant temperature to obtain an egg white mixture; (4) Adjust the pH of the egg white mixture, stir, and perform dynamic high-pressure microjet treatment to expose BCAA-related enzyme cleavage sites; (5) Enzymatic hydrolysis with complex protease to obtain an enzymatic hydrolysate by cutting BCAA-rich peptide segments; (6) Ultrafiltration of the enzymatic hydrolysate using an ultrafiltration membrane to remove large molecular impurities that are not completely hydrolyzed; (7) Passing the ultrafiltration permeate through a composite membrane assembly in sequence to separate BCAA to obtain an eluate; (8) Low-temperature vacuum spray drying of the eluate; and completing the preparation of the muscle growth egg white protein powder.

[0006] Preferably, the egg white stirring conditions in step (1) are 100-200 rpm stirring for 10-15 min; In step (2), the ultrasonic-shearing treatment conditions are: first ultrasonic treatment and then shearing treatment, 4-10℃ standing for 3-4 min after each treatment, and then cyclic ultrasonic-shearing treatment, a total of 2-3 cycles; the ultrasonic conditions are: power 30-50 W, treatment time 5-10 min, and the shearing conditions are: rate 3000-7000 rpm, treatment time 5-10 min.

[0007] Preferably, in step (3), the volume ratio of deflocculated egg white to deionized water is 1:(1-3); the prepared yeast β-glucan composite microspheres are added in a proportion of 0.5%-1.5% of the mass of the deflocculated egg white, and stirred at 25-30℃ for 5-10 min.

[0008] Preferably, in step (3), the preparation method of the yeast β-glucan composite microspheres is as follows: (a) Select yeast β-glucan and tea polyphenol to obtain mixed raw materials; (b) Add the mixed raw materials to deionized water, ultrasonically disperse under water bath to form a uniform suspension, add sodium alginate, and shear to obtain an emulsion; (c) Crosslink the above emulsion to form yeast β-glucan composite microspheres.

[0009] Preferably, in the step (3), in the step (a), the molecular weight of the yeast β-glucan is 10-50 kDa, and the mass ratio of the yeast β-glucan to the tea polyphenol is (3-5):1; In the step (b), the ultrasonic treatment is performed at 30-40℃ for 5-15 min at 30-40 W, the amount of the added sodium alginate is 0.3%-0.5% of the mass of the suspension, and the shearing condition is 4000-5000 rpm for 5-15 min; In the step (c), the cross-linking is performed at 25-35℃ for 15-20 min to form the yeast β-glucan composite microspheres with a particle size of 100-300 nm.

[0010] Preferably, in the step (4), the pH of the egg white mixture is adjusted to 3-4, and the stirring is performed at 4000-6000 rpm for 10-15 min; the dynamic high-pressure microfluidization treatment conditions are as follows: 4-5℃, 100-200 MPa, the material flow rate is controlled to make the single-pass time 0.1-0.5 s, and the circulation is performed 1-3 times, and the total treatment time is 0.3-1.5 s. The controllable unfolding of the egg white protein molecular structure exposes the originally embedded enzyme cutting sites.

[0011] Preferably, in the step (5), in the complex protease, the alkaline protease, the flavor protease, and the leucine aminopeptidase are composed, and the total enzyme activity is 800-2000 U / g based on the egg white protein, wherein the alkaline protease is 400-1000 U / g, the flavor protease is 200-500 U / g, and the leucine aminopeptidase is 200-500 U / g; the enzymolysis is performed at 40-70℃ for 2-3 h to target cut the BCAA-rich peptide segment; Preferably, in the step (6), the 8-14 kDa ultrafiltration membrane is used to continuously ultrafiltrate the enzymolysis liquid for 24-48 h to remove the unenzymolyzed protein and macromolecular impurities; at this time, the β-glucan-polyphenol composite microspheres are aggregated due to the neutral environment and are removed with the retentate, and no additional removal step is needed; In the step (8), the low-temperature vacuum spray drying conditions are as follows: the inlet air temperature is 120-180℃, the outlet air temperature is 50℃, the drying temperature is 5-10℃, and the vacuum degree is 0.1-0.2 mPa.

[0012] Preferably, in the step (7), the ultrafiltration liquid is subjected to gradient pH combined with an ultrafiltration-loose nanofiltration-enzymolysis-nanofiltration desalination composite membrane assembly, and the specific parameters are as follows: The first-stage ultrafiltration has a pore size of 5-10 kDa and a pH of 3.5-4.5, and the BCAA-rich peptide is retained; Second stage loose nanofiltration: the retention pore size is 300 Da, the pH of the first stage ultrafiltration retention liquid is adjusted to 6.5, and the BCAA-rich peptides are further concentrated; Enzymatic conversion: the second stage membrane is eluted by using a pH 7.5-8.5, NaHCO3 and NaCl buffer solution for charge shielding; the eluent is added with equal mass of flavor protease and protamex, and hydrolysis is carried out at 50-55 DEG C for 4-5 h, and the enzyme is inactivated at 90-95 DEG C for 15-20 min, so that the BCAA-rich peptides are completely hydrolyzed into high-activity BCAA-rich hydrolysate; Third stage nanofiltration desalination: the retention pore size is 80-90 Da, the free BCAA hydrolysate is adjusted to pH 7.0-7.2 and a conductivity <2 mS cm -1 Post-feeding, synchronous desalination and enrichment are realized.

[0013] Further preferably, the first stage ultrafiltration: the retention pore size is 5 kDa, the pH is 3.5-4.5, the BCAA-rich peptides (200-1000 Da) are retained due to the size being larger than the membrane pore size, and free amino acids, inorganic salts and pigments are discharged with the permeate, and the peptide retention rate is >90 %; Second stage loose nanofiltration: the retention pore size is 300 Da, the pH of the first stage ultrafiltration retention liquid is adjusted to 6.5, and the BCAA-rich peptides are further concentrated, and the impurity peptides and inorganic salts are more easily permeated, and the target peptide recovery rate is >85 %; Enzymatic conversion: the second stage membrane is eluted by using a pH 8.5, NaHCO3 and NaCl buffer solution for charge shielding; the eluent is added with equal mass of flavor protease and protamex (50 DEG C, pH 8.5, 1 % E / S, E / S is the mass ratio of enzyme to substrate), and hydrolysis is carried out at 50 DEG C for 4 h, and the enzyme is inactivated at 90 DEG C for 15 min, so that the BCAA-rich peptides are completely hydrolyzed into high-activity BCAA-rich hydrolysate; Third stage nanofiltration desalination: NF-90 Da, 0.02 m², TMP 20 bar, 25 DEG C, flux 12 L m -2 h -1 : the free BCAA hydrolysate is adjusted to pH 7.0 and a conductivity <2 mS cm - ¹Post-feeding; the free BCAA is partially permeated through the membrane due to low molecular weight and near electric neutrality, and Na + / Cl - Retention rate >80 %, synchronous desalination and enrichment are realized.

[0014] The egg white protein powder prepared by the preparation method of the muscle-increasing egg white protein powder is used for the preparation of health foods, special medical foods or drugs for repairing muscle damage and increasing muscle mass.

[0015] The present application has the following beneficial effects: 1、The present application selects the optimal composite protease ratio, which can specifically cut the egg white protein peptide bond to release branched-chain amino acid polypeptide, significantly increase the content of free leucine, and ensure the efficient combination of BCAA in the active structure of free end + hydrophobic side chain; on the process level, the low-temperature-short-time-continuous technology combination is adopted, the enzyme cutting site is accurately exposed by dynamic high pressure microjet pretreatment, and the enzyme hydrolysis efficiency is improved; combined with enzyme membrane reactor, BCAA is separated and continuously enriched in situ; then, the particles are homogenized and refined by ultrasonic cavitation shearing, and the temperature and oxygen partial pressure in the processing process are controlled by low-temperature vacuum spray drying, so that the oxidation and conformation change of BCAA are minimized. The egg white protein powder with high BCAA retention rate and low leucine oxidation rate is prepared, the muscle synthesis signal activation ability at cell level is significantly improved, not only providing an efficient and safe muscle building option for fitness people, but also enriching the preparation of domestic BCAA-rich egg white protein powder, and providing a new research idea and application direction for the field of food nutrition.

[0016] 2、The synergistic effect of ultrasonic-shear cycle treatment in egg white pretreatment stage and yeast beta glucan-polyphenol composite microspheres makes the dispersion degree of egg white protein increase, the microspheres are specifically combined with the protein to protect the BCAA fragments, and the selective enzyme cutting in the later enzyme hydrolysis is realized; the low-temperature coupling treatment of acid-dynamic high pressure microjet avoids the oxidation of BCAA and improves the exposure rate of enzyme cutting site. Yeast beta-glucan and tea polyphenols replace chemical protective groups, and the microspheres are naturally removed with the retentate in the neutral ultrafiltration stage, the product is free of any chemical residues, the residual amount of beta-glucan is less than 0.1%, which meets the strict standard of GB 2760; the polyphenol components in the composite microspheres give the product additional antioxidant activity, the in-vitro DPPH free radical scavenging ability is improved compared with ordinary egg white protein powder, and the dual functions of muscle building nutrition and antioxidant are realized.

[0017] 3、The composite microspheres are combined with low-temperature enzyme hydrolysis and vacuum drying to control the oxidation rate of BCAA; the gradient pH membrane separation technology accurately retains the non-target peptide segments, and the enrichment purity of BCAA is improved by 25% compared with single ultrafiltration, and the BCAA content of the finished product reaches more than 26%.

[0018] 4、The solubility of the product reaches 92.56%, and there is no precipitation and stratification in various food systems such as neutral beverages and milk-based protein powder, which is suitable for various sports nutrition food processing; the in-vitro simulated digestion BCAA release rate is more than 91%, which can be quickly absorbed and utilized by the human body, the muscle injury mouse experiment shows that the exhaustive exercise time is prolonged by 38% compared with the ordinary protein powder group, and the muscle repair efficiency is improved by 40%, which meets the efficient nutritional needs of fitness and special medical food.

[0019] 5. The BCAA-enriched egg white protein powder prepared by this invention can be widely compounded and applied in the fields of sports nutrition foods and foods for special medical purposes. It fills the technological gap in the green, efficient, and bioactive preparation of BCAA-enriched egg white protein in China, providing new research ideas and technical pathways for the functionalization of food, and possesses both significant scientific value and market application potential. Attached Figure Description Figure 1 The effect of different processing methods on the sensory evaluation of egg white protein; Figure 2 The effects of egg white protein powder under different treatments on the basic physiological indicators of mice with muscle injury; Figure 3 The effect of egg white protein powder on suspension time in mice with muscle injury under different treatment methods; Figure 4 The effect of egg white protein powder under different treatments on swimming exhaustion time in mice with muscle injury; Figure 5 The effects of egg white protein powder under different treatments on serum biochemical indicators in mice with muscle injury; Figure 6 The effects of egg white protein powder on gastrocnemius muscle in mice with muscle injury under different treatment methods; Figure 7 Effects of egg white protein powder on H&E staining in mice with muscle injury. Detailed Implementation

[0020] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0021] Explanation of the comparative measurement methods and results of the above embodiments: 1. Determination of solubility Bovine serum albumin was used as the standard protein. 8 mL of protein sample (1 mg / mL) was centrifuged at 10000 × g for 10 min. Then, 4 mL of biuret reagent was added to 1 mL of the supernatant and 1 mL of the uncentralized protein sample, respectively. After thorough mixing, the mixture was reacted in a dark room at room temperature for 30 min, and the absorbance was measured at 540 nm. The protein solubility expression is as follows: (1); In the formula, S and C a C b The values ​​are the protein solubility (%), supernatant protein concentration (mg / mL), and total protein concentration (mg / mL) of the egg white protein powder sample, respectively. 2. Turbidity Measurement The diluted protein sample (1 mg / mL) was incubated at room temperature for 30 min, and the absorbance at 660 nm was measured using a UV-Vis spectrophotometer. (Using A...) 660 This indicates the turbidity of the protein sample.

[0022] 3. Determination of surface hydrophobicity (H0) A mixture of 1 mL NaH₂PO₄ / Na₂HPO₄ buffer (0.6 mol / L NaCl, pH 6.5) and 200 μL bromophenol blue solution (1 mg / mL) was used as a control. First, 1 mL of protein sample (2 mg / mL) was mixed thoroughly with 200 μL bromophenol blue solution (1 mg / mL), then incubated at room temperature for 2 h, and centrifuged at 6000 ×g for 15 min. 0.5 mL of the supernatant was diluted to 4.5 mL of NaH₂PO₄ / Na₂HPO₄ buffer (0.6 mol / L NaCl, pH 6.5), and the absorbance was measured at 595 nm. The surface hydrophobicity of the protein was expressed as the amount of bromophenol blue binding to the protein, calculated as follows:

[0023] In the formula, A1 and A2 are the absorbance values ​​of the control and egg white protein powder samples, respectively.

[0024] 4. Particle size and potential determination Particle size distribution (PSD) was determined using a Malvern Mastersizer 2000 with a Scirocco 2000 dry sampler. The sample volume was adjusted to an opacity of 5–15%, and the results were repeated three times and the average value was taken. The zeta potential was measured at 0.1 mg / mL. - ¹The protein was dispersed in 0.1 mol / L solution. - ¹Phosphate buffer (pH 7.2), filtered through a 0.45 µm membrane and injected into a Zetasizer Nano-ZS, was measured three times at 25 °C.

[0025] 5. In vitro digestion simulation of protein powder According to INFOGEST standards, 40 mg of protein was administered orally sequentially (α-amylase 75 U mL). - ¹, pH 7, 2 min), stomach (pepsin 2000 U mL) - ¹, pH 3, 2 h) and intestinal (trypsin 100 µM + bile salts 10 mM, pH 7, 2 h) three-phase static digestion, terminated with 1 M NaOH and 5 mM Pefabloc SC respectively, centrifuged, the supernatant was lyophilized and dried. Store at 20 °C.

[0026] 6. Determination of leucine content Weigh a certain amount of egg white protein powder and dissolve it in deionized water to a concentration of 10 mg / mL. Take 2 mL of this solution, add 3 mL of ninhydrin hydrate solution and 0.1 mL of 0.3% ascorbic acid solution, mix well, boil in boiling water for 15 min, then cool in cold water and shake well. Take 5 mL of the sample extract and centrifuge at 8000 r / min for 10 min. Measure the absorbance of the supernatant at a wavelength of 580 nm. Prepare a standard substance using leucine, and plot a standard curve with absorbance as the ordinate and concentration as the abscissa. The sample content can be read from the standard curve. 7. Determination of Amino Acid Content in Protein Powder The amino acid content in the sample was determined according to GB / T 5009.124—2003, "Determination of Amino Acids in Food". 0.1 g (accurate to 0.0001 g) of egg white protein was placed in a hydrolysis tube. The hydrolysis solution was 10 mL of 6 mol / L hydrochloric acid solution. The hydrolysis temperature was 115 ℃, and the hydrolysis time was 24 h. After cooling, the solution was diluted to volume in a 50 mL volumetric flask. 4 mL of the diluted solution was poured into an evaporating dish and evaporated to dryness in a 90 ℃ water bath. The sample was dissolved in 0.02 mol / L hydrochloric acid and diluted to 20 mL. The test solution was filtered through a 0.22 μm filter membrane and analyzed using an amino acid analyzer. The injection volume was 20 μL, and the amino acid content in the sample was determined.

[0027] 8. Sensory evaluation An evaluation panel consisting of 20 food science graduate students was formed. Before the evaluation, they received unified training and conducted sensory evaluations of the flavor and taste of the egg white protein powder after reconstitution, according to Table 1.

[0028] Table 1 Sensory Evaluation Form

[0029] 9. Animal Experiment Design ICR grade male mice (6-8 weeks old) were acclimatized for 7 days in a standard light / dark cycle with free access to food and water. They were then divided into 6 groups: normal diet group (control group), muscle injury model group (model group), Example 1 group, Comparative Example 1 group, Comparative Example 5 group, and Comparative Example 6 group. The model group was injected with 50 μL of 0.5% bupivacaine hydrochloride injection into the right leg of the mice to establish a mouse model of muscle injury. Changes in mouse weight were recorded. After 14 days of continuous feeding, the mice were quickly sacrificed by cervical dislocation, and the mouse muscle tissue was collected for subsequent testing and analysis. 10. Determination of mouse suspension time Muscle strength was assessed using a limb suspension test. To ensure the test animals were fully awake, mice were brought into the laboratory 20 minutes before the experiment and placed in the center of a wire 40 cm above the mat. The suspension time of the limbs, used to demonstrate muscle strength, was recorded until the mice were released. 11. Measurement of Exhaustion Swimming Time The exhaustive swimming test was used to evaluate the motor ability of mice. Simply put, 30 minutes after the last gavage, the mice's tails were attached to a wire (5% of the mouse's body weight), and a cubic swimming pool (70 cm × 80 cm × 80 cm) was filled with warm water at 30 ± 3 ℃. The endurance performance of each group of mice was assessed based on the swimming time. The time from the start of swimming to exhaustion was recorded. When a mouse failed to swim to the surface within 5 seconds after entering the water, it was considered exhausted.

[0030] 12. Determination of physiological and biochemical indicators in mice Blood samples were collected from the orbital venous sinuses of mice, and after standing at room temperature for 2 hours, mouse serum was obtained by centrifugation at 3000 g for 10 minutes. The levels of blood urea nitrogen, creatine kinase, and lactate dehydrogenase were measured using a biochemical analyzer (Hitachi, Japan).

[0031] 13 H&E staining Mouse gastrocnemius muscle was immersed in paraffin, cut into 10 μm thick sections, stained with hematoxylin and eosin, and then the tissue morphology was observed using an optical microscope.

[0032] Example 1 A novel method for preparing egg white protein muscle-building powder includes the following steps: (1) Egg white separation: Select fresh eggs, disinfect the eggshells with 75% ethanol, crack the shells, and separate the egg whites from the yolks; stir the egg whites at 150 rpm for 12 min to make the egg white system uniform; (2) The egg white was subjected to ultrasonic-shear treatment to obtain deflocculated egg white; (3) Add deionized water to the deflocculated egg white to dilute it, add yeast β-glucan complex microspheres, stir at a constant temperature to obtain egg white mixture; (4) Adjust the pH of the egg white mixture, stir, and perform dynamic high-pressure microjet treatment to expose BCAA-related enzyme sites; (5) Enzymatic hydrolysis was performed using a complex protease to cut BCAA-rich peptides and obtain the hydrolysate. (6) Use an ultrafiltration membrane to ultrafilter the enzymatic hydrolysate to remove macromolecular impurities that are not completely hydrolyzed; (7) The ultrafiltration permeate is passed through the composite membrane module in sequence to separate BCAA and obtain the eluent; (8) The eluent is spray-dried at low temperature under vacuum; the preparation of muscle-building egg white protein powder is completed.

[0033] In step (2), the ultrasonic shearing treatment conditions are: ultrasonic treatment is performed first, followed by shearing treatment. After each treatment, the sample is left to stand at 4°C for 4 minutes, and then the ultrasonic shearing treatment is repeated 3 times. The ultrasonic conditions are: power 40 W, treatment for 6 minutes. The shearing conditions are: speed 7000 rpm, treatment for 6 minutes.

[0034] In step (3), the volume ratio of deflocculated egg white to deionized water is 1:2; the prepared yeast β-glucan composite microspheres are added at a ratio of 0.5% of the mass of the flocculated egg white, and stirred at 25°C for 8 minutes.

[0035] The preparation method of yeast β-glucan complex microspheres is as follows: (a) Yeast β-glucan and tea polyphenols were mixed to obtain a mixed raw material; (b) Add the mixed raw materials to deionized water, disperse them by ultrasonication in a water bath to form a homogeneous suspension, add sodium alginate, and shear to obtain an emulsion; (c) The above emulsion is allowed to stand and crosslink to form yeast β-glucan composite microspheres.

[0036] In step (3), in step (a), the molecular weight of yeast β-glucan is 10-50 kDa, and the mass ratio of yeast β-glucan to tea polyphenols is 4:1; In step (b), the sodium alginate is added at 30°C and 30W for 5 min, the amount of sodium alginate added is 0.4% of the mass of the suspension, and the shearing condition is 4000 rpm for 5 min. In step (c), cross-linking is performed at 25°C for 15 min to form yeast β-glucan composite microspheres with a particle size of 100 nm.

[0037] In step (4), the pH of the egg white mixture is adjusted to 3.5, and the mixture is stirred at 5000 rpm for 12 min. The dynamic high-pressure microjet treatment conditions are: 4 ℃, 100 MPa, and the material flow rate is controlled so that the single pass time is 0.2 s, and the process is repeated 3 times. This allows the egg white protein molecular structure to unfold in a controlled manner, exposing the enzyme cleavage sites that were originally embedded within it.

[0038] In step (5), the complex protease is composed of alkaline protease, flavor protease and leucine aminopeptidase, with a total enzyme activity of 1000 U / g based on egg white protein, including 400 U / g of alkaline protease, 400 U / g of flavor protease and 200 U / g of leucine aminopeptidase; it is enzymatically hydrolyzed at 50℃ for 2.5h to target and cleave BCAA-rich peptides. In step (6), the enzymatic hydrolysate is continuously ultrafiltered for 30 h using a 10 kDa ultrafiltration membrane to remove undigested proteins and macromolecular impurities. At this time, the β-glucan-polyphenol composite microspheres aggregate due to the neutral environment and are removed with the retentate, without the need for additional removal steps. Step (7) gradient pH membrane separation and purification specifically includes the following operations: primary ultrafiltration (UF-5 kDa, PES (polyethersulfone), 0.1 m 2 Transmembrane pressure TMP 0.8 bar, 25°C, flux 60 L m -2 h -1 ): Add 0.2 mol L online -1 The pH was adjusted to 3.5-4.5 with HCl. BCAA-rich peptides (200-1000 Da) were retained because their size was larger than the membrane pore size. Free amino acids, inorganic salts, and pigments were discharged with the permeate. The peptide retention rate was >90%. The second stage was loose nanofiltration (NF-300 Da, polyamide composite membrane, 0.05 μm). 2 TMP 12 bar, 25 °C, flux 18 L m -2 h -1 ): Use 0.2 mol L of retentate. -1 Adjusting the NaOH concentration to pH 6.5, the positively charged BCAA-rich peptides undergo Donnan repulsion against the negatively charged membrane surface, resulting in further concentration. Impurities and inorganic salts permeate more easily, achieving a target peptide recovery rate >85%. Enzymatic hydrolysis: using pH 8.5, 0.1 mol / L... -1 NaHCO3 and 0.15 mol L -1 The second-stage membrane was eluted with charge-shielded NaCl buffer (3 × 100 mL); the eluent was then mixed with equal masses of flavor protease and protease (50 °C, pH 8.5, 1% E / S, where E / S is the enzyme-to-substrate mass ratio), hydrolyzed at 50 °C for 4 h, and then inactivated at 90 °C for 15 min to completely hydrolyze the BCAA-rich peptides into free BCAA (130-147 Da); the third-stage nanofiltration desalting was performed (NF-90 Da, 0.02 m², TMP 20 bar, 25 °C, flux 12 Lm). -2 h -1 The hydrolysate was adjusted to pH 7.0 and had a conductivity of <2 mS / cm. - ¹Post-feed; Free BCAA, due to its low molecular weight and near-neutral charge, partially permeates the membrane, while Na… + / Cl - With a retention rate of >80%, simultaneous desalination and enrichment are achieved.

[0039] (8) Finished product preparation: Low-temperature vacuum spray drying; complete the preparation of muscle-building egg white protein powder. The low-temperature vacuum spray drying conditions are: inlet air temperature 160℃, outlet air temperature 50℃, drying temperature 8℃, and vacuum degree 0.15 mPa.

[0040] Example 2 Following the steps of Example 1, only the total enzyme activity of the complex protease was adjusted to 1200 U / g, including 400 U / g of alkaline protease, 400 U / g of flavor protease, and 400 U / g of leucine aminopeptidase. Other process parameters and operations remained unchanged.

[0041] Example 3 Following the steps of Example 1, only the total enzyme activity of the complex protease was adjusted to 1800 U / g, including 600 U / g of alkaline protease, 600 U / g of flavor protease, and 400 U / g of leucine aminopeptidase. Other process parameters and operations remained unchanged.

[0042] Example 4 Following the steps of Example 1, only the dynamic high-pressure microjet treatment pressure is adjusted to 80 MPa, while other process parameters and operations remain unchanged.

[0043] Example 5 Following the steps of Example 1, only the dynamic high-pressure microjet processing pressure is adjusted to 120 MPa, while other process parameters and operations remain unchanged.

[0044] Comparative Example 1 (without composite microspheres, otherwise the same as Example 1) Referring to the steps of Example 1, except for the addition of composite microspheres in step (3), other process parameters and operations remain unchanged.

[0045] Comparative Example 2 (single protease, otherwise the same as Example 1) Referring to the steps of Example 1, in step (5), only alkaline protease (enzyme activity 1000 U / g) is used to replace the complex protease, while other process parameters and operations remain unchanged.

[0046] Comparative Example 3 (Binary complex protease: alkaline protease + flavor protease, the rest is the same as in Example 1) Referring to the steps of Example 1, only the complex protease in step (5) is adjusted to a binary combination of alkaline protease and flavor protease = 2:1, and the total enzyme activity is kept at 1000 U / g (calculated based on egg white protein content, alkaline protease 400 U / g and flavor protease 600 U / g), while other process parameters and operations remain unchanged.

[0047] Comparative Example 4 (leucine aminopeptidase missing: alkaline protease + flavor protease + neutral protease, the rest is the same as in Example 1) Referring to the steps of Example 1, only the complex protease in step (6) is adjusted to a combination of alkaline protease: flavor protease: neutral protease (neutral protease is used to replace leucine aminopeptidase), the total enzyme activity is kept at 1000 U / g (based on egg white protein content), and other process parameters and operations remain unchanged.

[0048] Comparative Example 5 (without dynamic high-pressure microjets, otherwise the same as Example 1) Referring to the steps of Example 1, the dynamic high-pressure microjet treatment in step (4) is deleted, while other process parameters and operations remain unchanged.

[0049] Comparative Example 6 (Traditional chemical protective agent process, no innovation) Referring to the steps of Example 1, in step (3), 0.5% sodium sulfite (chemical protectant) is used to replace the composite microspheres, and the dynamic high-pressure microjet treatment in step 2 is deleted. Other process parameters and operations remain unchanged.

[0050] Comparative Example 7 (no gradient pH membrane separation, only single ultrafiltration, the rest is the same as Example 1) Referring to the steps in Example 1, the gradient pH membrane separation and purification step in step 7 is removed, and only the permeate from the 15 kDa ultrafiltration membrane is directly dried, while other process parameters and operations remain unchanged.

[0051] Performance testing Physicochemical property testing The solubility, turbidity, and surface hydrophobicity of the egg white protein peptides prepared in Examples 1-5 and Comparative Examples 1-7 were tested using the following methods: Table 2

[0052] The solubility of a protein determines its ability to maintain its native conformation and functional activity in solution. Table 1 shows that different treatment methods resulted in significant differences in the solubility of egg white protein. Table 1 also shows that Example 1 exhibited the highest solubility (92.56 ± 0.56%), the lowest turbidity (1.20 ± 0.42%), and the best surface hydrophobicity (65.25 ± 1.23%), significantly outperforming all the comparative examples. Comparative Example 1 (without composite microspheres) had a solubility of 86.53±0.57% due to the lack of dispersion and protection by microspheres; Comparative Example 5 (without dynamic high-pressure microjets) had a solubility of only 80.65±1.78% due to insufficient exposure of enzyme cleavage sites and inadequate protein particle refinement; Comparative Example 6 (chemical protectant) had a solubility of 88.42±0.74%, but this was lower than that of Example 1, and there was a risk of chemical residue; Comparative Example 3 (binary composite enzyme) and Comparative Example 4 (leucine-deficient aminopeptidase) had solubilities of 82.15±0.63% and 81.32±0.58%, respectively, which were lower than those of Example 1 due to insufficient enzymatic hydrolysis efficiency and incomplete protein degradation. This indicates that the synergistic effect of composite microsphere dispersion and dynamic high-pressure microjet refinement can significantly improve the dispersibility and stability of egg white protein. At the same time, the efficient enzymatic hydrolysis of the ternary composite protease system lays the foundation for optimizing protein solubility performance and ensures the structural integrity and bioactivity of branched amino acid peptides throughout the entire processing chain, providing a basis for subsequent high-value applications.

[0053] Colloid stability test The egg white protein peptides prepared in Examples 1-5 and Comparative Examples 1-7 were subjected to ζ-potential and particle size tests, and the test methods are as follows: ζ-potential: The sample was dissolved in deionized water to prepare a 0.1% solution using a Malvern laser particle size analyzer and measured at 25°C. Particle size: The volume average particle size was measured using a Malvern laser particle size analyzer. The sample was dissolved in deionized water to prepare a 0.1% solution, and the volume average particle size was measured at 25°C.

[0054] The test results are shown in Table 2.

[0055] Table 3

[0056] Example 1 has the smallest particle size (855.25±65.23 nm) and the largest absolute value of ζ-potential (-43.67±0.62 mV), indicating that it has the best colloidal dispersion stability. Comparative Example 5 (without dynamic high-pressure microjets) had an increased particle size of 3790.52±89.12 nm and a decreased potential of -25.91±0.12 mV due to insufficient protein structure expansion. Comparative Example 1 (without composite microspheres) had an increased particle size of 3760.53±180.65 nm due to the lack of steric stabilization from the microspheres. Comparative Examples 3 (binary composite enzyme) and 4 (leucine-deficient aminopeptidase) had incomplete enzymatic hydrolysis and incomplete protein particle degradation, with particle sizes of 3125.68±78.45 nm and 3256.74±89.36 nm, respectively, and absolute potential values ​​of -33.25±0.42 mV and -32.18±0.35 mV, respectively, all of which were inferior to Example 1. This confirms that dynamic high-pressure microjets can effectively refine protein particles, and the composite microspheres can enhance system stability and protect BCAA fragments through charge repulsion. Simultaneously, the efficient enzymatic hydrolysis by the ternary complex protease further optimizes the particle size distribution and dispersion stability of the protein particles. A high negative potential signifies increased system density and enhanced electrostatic repulsion, effectively inhibiting hydrophobic aggregation and maintaining colloidal dispersion stability. Ultimately, the smallest particle size (855.25 nm) and the highest potential were observed. Example 1 (43.67 mV) boosted solubility to 92%, achieving synergistic optimization of particle size-potential-solubility.

[0057] BCAA content and in vitro release test Leucine, as the rate-limiting activator of branched-chain amino acids (BCAAs), directly initiates muscle protein synthesis via the mTOR-C1 signaling axis. Table 4 shows that different treatment processes resulted in significant gradients in leucine content in egg white protein powder. Example 1 showed the highest BCAA content (26.67 ± 0.35%) and a leucine content of 89.34 ± 0.56 mg / g prot, significantly higher than the control groups. Comparative Example 2 (single protease) had a BCAA content of only 19.31±0.31% due to the lack of synergistic cleavage; Comparative Example 1 (without composite microspheres) had a BCAA content of 19.47±0.26% due to BCAA oxidation loss; Comparative Example 5 (without dynamic high-pressure microfluidic) had a BCAA content of only 19.48±0.14% due to low enzymatic hydrolysis efficiency; Comparative Example 6 (chemical protectant) had a BCAA content of 22.67±0.26%, but this was lower than that of Example 1, and there was a risk of chemical residue; Comparative Example 3 (binary composite enzyme) had a BCAA content of only 21.35±0.28% due to the lack of targeted release of leucine aminopeptidase; Comparative Example 4 (leucine aminopeptidase missing, replaced by neutral protease) had a BCAA content of only 20.86±0.32% because neutral protease could not accurately cleave the characteristic peptide bonds of BCAA, all of which were significantly lower than that of Example 1. This indicates that the combination of synergistic cleavage by complex protease, activation by microspheres, and enhanced effect by microfluidics can achieve efficient enrichment and activity retention of BCAA. Furthermore, the addition of leucine aminopeptidase in the ternary complex system is the key to increasing BCAA content, showing significant advantages over binary complex enzymes, single proteases, and non-targeted ternary enzyme combinations.

[0058] The results of the in vitro release of branched-chain amino acids are shown in Table 4. Example 1 showed the highest in vitro BCAA release (15.56 ± 0.24%), a 32% increase compared to Comparative Example 2 (single protease), a 41% increase compared to Comparative Example 5 (without microfluidics), a 12% increase compared to Comparative Example 6 (chemoprotectant), a 20% increase compared to Comparative Example 3 (binary complex enzyme), and a 23% increase compared to Comparative Example 4 (leucine-deficient aminopeptidase). This indicates that the ternary complex protease system of the present invention can significantly improve the enzymatic hydrolysis efficiency and bioavailability of BCAA. The targeting action of leucine aminopeptidase promotes the efficient release of BCAA from peptides, ensuring its rapid absorption and utilization during human digestion, and providing sufficient active amino acids for muscle synthesis.

[0059] Table 4

[0060] Sensory evaluation of egg white protein powder Sensory evaluation of egg white protein powder under different processing conditions, such as Figure 1As shown in the figure, the sensory evaluation of egg white protein powder mainly reflects five aspects: umami, sweetness, bitterness, astringency, and fishy taste. Example 1 showed the lowest bitterness, astringency, and fishy taste values, and also possessed a certain degree of sweetness, indicating that the protein powder of Example 1 had the best flavor and was more likely to be accepted by consumers. Considering the solubility of egg white protein, the enrichment level of leucine, and the sensory evaluation of egg white protein, Example 1, Comparative Example 1, Comparative Example 5, and Comparative Example 6 were ultimately selected for subsequent mouse muscle-building effect evaluation experiments. Effects of egg white protein powder on basic physiological indicators in mice with muscle injury The effect of egg white protein powder on body weight in mice with muscle damage, such as Figure 2 As shown. By Figure 2 It can be seen that the model group was lighter than the control group after the feeding period. After protein intake, the weight loss trend of mice was gradually reversed, but the egg white protein powders of different treatments had different reversal abilities. Among them, the egg white protein powder prepared in Example 1 had the best ability to reverse the weight loss of mice. This may be due to the fact that Example 1 has more branched-chain amino acids, especially leucine.

[0061] Effect of egg white protein powder on suspension time in mice with muscle injury The effect of egg white protein powder on suspension time in mice with muscle injury, such as Figure 3 As shown in the figure, the suspension time in the model group was shorter compared to the control group. However, after egg white protein intake, the suspension time increased, with Example 1 showing a slightly greater increase in suspension time than the other groups. This may be because adequate long-term protein intake can increase muscle volume, and the increase in muscle volume is related to the increase in strength. In addition, protein intake helps reduce muscle protein breakdown after exercise, protecting muscle mass. Example 1 showed the best alleviating effect, which may be due to the production of more branched-chain amino acids through multi-step processing. These branched-chain amino acids are direct precursors for muscle protein synthesis.

[0062] Effect of egg white protein powder on exhaustive swimming time in mice with muscle injury The effect of egg white protein powder on the exhaustive swimming time of mice with muscle damage, such as Figure 4 As shown. By Figure 4 It was found that the model group had a shorter time to exhaustion compared to the control group. However, after egg white protein intake, the time to exhaustion increased, with Example 1 showing a slightly greater increase in the time to exhaustion than the other groups. This may be because adequate long-term protein intake can increase muscle volume, and the increase in muscle volume is related to the increase in strength. In addition, protein intake helps reduce muscle protein breakdown after exercise, protecting muscle mass.

[0063] Effects of egg white protein powder on biochemical indicators in mice with muscle injury Measuring creatine kinase (CK) and lactate dehydrogenase (LDH) levels is of significant research value for assessing the degree of muscle injury in mice, monitoring the recovery process, evaluating treatment efficacy, and gaining a deeper understanding of the pathophysiological mechanisms of muscle injury. Blood urea nitrogen (BUN) is an important indicator for assessing renal excretion function, as it is primarily excreted through the kidneys. Monitoring BUN levels in cases of muscle injury helps assess kidney health. The biochemical indicators of egg white protein powder in mice with muscle injury include... Figure 5 As shown in the figure, compared with the control group, the levels of CK, LDH, and BUN in the model group were significantly increased. This may be because when muscle cells are damaged, CK, LDH, and BUN are released from the cells into the bloodstream, resulting in elevated serum levels. This also indicates severe muscle damage. After protein powder intake, the above values ​​decreased significantly, and the above trends were reversed. Although there were no significant differences among the groups, the levels in Example 1 were the lowest.

[0064] Effects of egg white protein powder on gastrocnemius muscle mass in mice with muscle damage Measuring gastrocnemius muscle mass in mice is an important tool for assessing muscle injury, function, molecular mechanisms, treatment effects, and muscle aging, and is of great significance for research on mice with muscle injury. Furthermore, measuring gastrocnemius muscle mass helps monitor the dynamic process of muscle injury and recovery. The effect of protein powder on gastrocnemius muscle mass in mice with muscle injury is shown in the figure. Figure 6 It was found that the gastrocnemius muscle mass of the model groups was reduced compared with that of the control group, which reflects a certain degree of muscle atrophy in the mice. The reduction in gastrocnemius muscle mass may be related to decreased protein synthesis and increased protein degradation. After protein intake, the mass of the gastrocnemius muscle of the mice increased, with Example 1 showing the best effect on increasing the mass of the gastrocnemius muscle of the mice, followed by Comparative Example 1, Comparative Example 5, and Comparative Example 6.

[0065] Effects of egg white protein powder on H&E staining of gastrocnemius muscle in mice with muscle injury Egg white protein powder on H&E staining of gastrocnemius muscle in mice with muscle damage, as shown Figure 7 As shown in the figure, compared with the control group, the model group showed a significantly greater increase in the number of inflammatory cells (inflammatory infiltration). After ingestion of egg white protein powder, the mouse gastrocnemius muscle exhibited a normal histological appearance with very few inflammatory cells. This also indicates that egg white protein under different treatment conditions has different muscle-building effects, with Example 1 showing the best muscle repair and growth effects, possibly due to the abundance of branched-chain amino acids, particularly leucine, in the egg white protein under this condition. These results are also consistent with the amino acid results mentioned above.

[0066] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The embodiments and features described in these embodiments can be arbitrarily combined without conflict. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A novel method for preparing egg white protein muscle-building powder, characterized in that, Includes the following steps: (1) Egg white separation: Select fresh eggs, disinfect the eggshells with ethanol, crack the shells, and separate the egg whites and yolks; stir the egg whites to make the egg white system uniform; (2) The egg white was subjected to ultrasonic-shear treatment to obtain deflocculated egg white; (3) Add deionized water to the deflocculated egg white to dilute it, add yeast β-glucan complex microspheres, stir at a constant temperature to obtain egg white mixture; (4) Adjust the pH of the egg white mixture, stir, and perform dynamic high-pressure microjet treatment to expose BCAA-related enzyme sites; (5) Enzymatic hydrolysis was performed using a complex protease to cut BCAA-rich peptides and obtain the hydrolysate. (6) Use an ultrafiltration membrane to ultrafilter the enzymatic hydrolysate to remove large molecular impurities that are not completely hydrolyzed; (7) Pass the ultrafiltration permeate through the composite membrane module in sequence to separate BCAA and obtain the eluent; (8) The eluent is spray-dried at low temperature under vacuum; the preparation of muscle-building egg white protein powder is completed.

2. The method for preparing the novel egg white protein muscle-building powder according to claim 1, characterized in that, The egg white stirring conditions in step (1) are: 100-200 rpm for 10-15 min; In step (2), the ultrasonic shearing treatment conditions are: ultrasonic treatment is performed first, followed by shearing treatment. After each treatment, the temperature is settling at 4-10℃ for 3-4 minutes, and then ultrasonic shearing treatment is repeated 2-3 times. The ultrasonic conditions are: power 30-50 W, treatment time 5-10 minutes. The shearing conditions are: speed 3000-7000 rpm, treatment time 5-10 minutes.

3. The method for preparing the novel egg white protein muscle-building powder according to claim 1, characterized in that, In step (3), the volume ratio of deflocculated egg white to deionized water is 1:(1~3); the prepared yeast β-glucan composite microspheres are added at a ratio of 0.5%-1.5% of the mass of the deflocculated egg white, and stirred at 25-30℃ for 5-10 min.

4. The method for preparing the novel egg white protein muscle-building powder according to claim 1, characterized in that, In step (3), the preparation method of yeast β-glucan composite microspheres is as follows: (a) Yeast β-glucan and tea polyphenols were mixed to obtain a mixed raw material; (b) Add the mixed raw materials to deionized water, disperse them by ultrasonication in a water bath to form a homogeneous suspension, add sodium alginate, and shear to obtain an emulsion; (c) The above emulsion is allowed to stand and crosslink to form yeast β-glucan composite microspheres.

5. The method for preparing the novel egg white protein muscle-building powder according to claim 1, characterized in that, In step (3), in step (a), the molecular weight of yeast β-glucan is 10-50 kDa, and the mass ratio of yeast β-glucan to tea polyphenols is (3~5):1; In step (b), the suspension is sonicated at 30-40 W for 5-15 min at 30-40℃, the amount of sodium alginate added is 0.3%-0.5% of the mass of the suspension, and the shearing condition is shearing at 4000-5000 rpm for 5-15 min. In step (c), cross-linking is performed at 25-35℃ for 15-20 min to form yeast β-glucan composite microspheres with a particle size of 100-300 nm.

6. The method for preparing the novel egg white protein muscle-building powder according to claim 1, characterized in that, In step (4), the pH of the egg white mixture is adjusted to 3-4, and the mixture is stirred at 4000-6000 rpm for 10-15 min. The dynamic high-pressure microjet treatment conditions are: 4-5 ℃, 100-200 MPa, and the material flow rate is controlled so that the single pass time is 0.1-0.5 s, and the mixture is circulated 1-3 times.

7. The method for preparing the novel egg white protein muscle-building powder according to claim 1, characterized in that, In step (5), the complex protease consists of alkaline protease, flavor protease, and leucine aminopeptidase, with a total enzyme activity of 800-2000 U / g based on egg white protein, including 400-1000 U / g of alkaline protease, 200-500 U / g of flavor protease, and 200-500 U / g of leucine aminopeptidase; it is enzymatically hydrolyzed at 40-70℃ for 2-3 h to cleave BCAA-rich peptides.

8. The method for preparing the novel egg white protein muscle-building powder according to claim 1, characterized in that, In step (6), the enzymatic hydrolysate is continuously ultrafiltered for 24-48 h using an 8-14 kDa ultrafiltration membrane; In step (8), the low-temperature vacuum spray drying conditions are: inlet air temperature 120-180℃, outlet air temperature 50℃, drying temperature 5-10℃, and vacuum degree 0.1-0.2 mPa.

9. The method for preparing the novel egg white protein muscle-building powder according to claim 1, characterized in that, In step (7), the ultrafiltrate is treated with a gradient pH combined with an ultrafiltration-loose nanofiltration-enzymatic hydrolysis-nanofiltration desalination composite membrane module, with the following specific parameters: First-stage ultrafiltration: pore size cutoff is 5-10 kDa, pH 3.5-4.5, BCAA-rich peptides are retained; Second-stage loose nanofiltration: pore size cutoff is 300 Da, pH of the first-stage ultrafiltration retentate is adjusted to 6.5, and BCAA peptides are enriched for further concentration; Enzymatic hydrolysis and conversion: The second-stage membrane was eluted with charge shielding using a pH 7.5-8.5, NaHCO3 and NaCl buffer solution; the eluent was then hydrolyzed with equal masses of flavor protease and protease at 50-55 ℃ for 4-5 h, and the enzymes were inactivated at 90-95 ℃ for 15-20 min. Third-stage nanofiltration desalination: pore size cutoff 80-90 Da, pH adjusted to 7.0-7.2 with free BCAA hydrolysate, conductivity <2 mS / cm. -1 Feeding later.

10. The egg white protein powder obtained by the preparation method of the novel egg white protein muscle-building powder according to any one of claims 1-9 is used to prepare health food, special medical food or medicine for repairing muscle damage and increasing muscle mass.

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