Alligator egg yolk lecithin polypeptide extraction method

Abstract

The application relates to the technical field of natural extracts, and discloses an extraction method of alligator egg yolk lecithin polypeptide, which comprises the following steps: preparing egg yolk dry powder by pulsed ultrasonic crushing and freeze-drying after slurry preparation of fresh alligator egg yolk; defatting the egg yolk dry powder by supercritical CO2 extraction, and then centrifuging the defatted residue after lecithin extraction by ethanol immersion, and obtaining alcohol-free protein residue by vacuum dealcoholization of the precipitate; obtaining polypeptide crude extract by complex enzymolysis of the alcohol-free protein residue; mixing the polypeptide liquid with lecithin liquid after ultrafiltration and microfiltration; and sequentially modifying the mixed liquid by sodium citrate, sucrose fatty acid ester, polyethylene glycol 400 and sodium alginate, and then concentrating and drying the modified mixed liquid to obtain a finished product. The application realizes the synergistic extraction of lecithin and polypeptide in alligator egg yolk, is beneficial to the alleviation of the problems of enzyme activity inhibition, membrane pollution and poor solubility of products, has good safety in the whole technological process, and has stable product composition.

Description

Technical Field

[0001] This invention relates to the field of natural extract technology, specifically a method for extracting lecithin polypeptides from crocodile egg yolk. Background Technology

[0002] With increasing attention being paid to the development of functional foods and special biological resources, the high-value utilization of by-products from crocodiles, as artificially bred economic animals, has certain development potential. Crocodile egg yolks contain lecithin and protein. Lecithin is related to the nutritional protection of the nervous system, and protein, after hydrolysis, can form polypeptides with potential activities such as antioxidation and immune regulation. Therefore, the synergistic extraction of lecithin and polypeptides from crocodile egg yolks has the basis for further development and utilization.

[0003] In current egg yolk processing methods, lecithin is typically obtained through organic solvent extraction, while peptides are mostly prepared via enzymatic hydrolysis. These methods are well-established for single-component preparation, but when used in complex matrices like crocodile egg yolk where lipids and proteins are tightly intertwined, they are easily limited by the raw material structure and subsequent separation processes.

[0004] Direct defatting with organic solvents carries the risk of solvent residue and has limited selectivity in removing lipid impurities such as cholesterol, potentially affecting the purity of subsequent lecithin products. Direct enzymatic hydrolysis of whole crocodile egg yolks can hinder sufficient contact between the protease and protein substrate, impacting hydrolysis efficiency. Even with a defatting-then-enzymatic hydrolysis approach, residual organic solvents from the defatting stage may inhibit subsequent protease activity, affecting the stability of the enzymatic hydrolysis process. If lecithin and peptides are directly mixed and separated after initial extraction, the micelles or lipid aggregates formed by lecithin can easily clog membrane pores and reduce flux, hindering the simultaneous purification of both components. Furthermore, the resulting mixture may exhibit poor dispersibility, insufficient solubility, or stratification in water due to the hydrophobicity of lecithin and some peptide fragments, limiting its application in liquid formulations.

[0005] Therefore, this invention proposes a method for extracting lecithin polypeptides from crocodile egg yolks to overcome the shortcomings of existing technologies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for extracting lecithin peptides from crocodile egg yolks, which solves the problems of low process efficiency and poor solubility of the final product when extracting lecithin and peptides from crocodile egg yolks.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for extracting lecithin polypeptides from crocodile egg yolks, comprising the following steps: S1. Fresh crocodile egg yolks are mixed with deionized water to make egg yolk homogenate, which is then broken by pulsed ultrasound and freeze-dried to obtain egg yolk powder. S2. The egg yolk powder is extracted with supercritical CO2 to obtain defatted residue; the defatted residue is extracted with ethanol and centrifuged, the supernatant is used as crude lecithin extract, and the precipitated residue is de-alcoholized under reduced pressure to obtain alcohol-free protein residue. S3. The alcohol-free protein residue is added to deionized water, and then hydrolyzed with alkaline protease and neutral protease in sequence. Phospholipase is added for modification, and the enzyme is inactivated by centrifugation to obtain a crude polypeptide extract. The crude polypeptide extract is subjected to microfiltration and ultrafiltration to obtain an ultrafiltration permeate, which is mixed with the microfiltration crude lecithin extract to obtain a lecithin polypeptide mixture. S4. Adjust the pH value of the lecithin polypeptide mixture, add sodium citrate and sucrose fatty acid ester and stir, add polyethylene glycol 400 under ultrasonication, add sodium alginate and stir to obtain a modified mixture; the modified mixture is concentrated under reduced pressure and freeze-dried to obtain the finished product.

[0008] By adopting the above technical solution, this invention establishes a set of interconnected process routes. The first is the pretreatment of raw materials. Through the cavitation effect and mechanical vibration of pulsed ultrasound, the dense emulsified particles composed of lipoproteins and phospholipids in the crocodile egg yolk matrix are physically destroyed under mild conditions, so that the protein and lipid components encapsulated inside can be exposed. The subsequent freeze drying sublimates water at low temperature, which not only helps to reduce the adverse effects of heat drying on proteins, but also obtains the dry powder state required for subsequent supercritical fluid extraction.

[0009] Building upon this foundation, to achieve effective separation of lipids and proteins, this method utilizes the selective solubility of nonpolar molecules by supercritical carbon dioxide to remove neutral lipid components such as cholesterol and triglycerides from egg yolk powder. This results in a relative enrichment of lecithin and protein in the residue, achieving pre-purification using carbon dioxide as the medium. For the separated protein residue, subsequent depressurized deethanolification is crucial for ensuring highly active enzymatic hydrolysis. The core of this process lies in lowering the boiling point of ethanol under low temperature and low pressure conditions, removing residual ethanol under milder conditions, and providing a suitable environment for subsequent protease catalysis.

[0010] One innovation of this method is the use of a split purification strategy. The hydrolysate containing small-molecule peptides and the ethanol extract containing lecithin micelles are processed separately. The crude peptide extract is ultrafiltered to retain unhydrolyzed macromolecules, while the crude lecithin extract only needs microfiltration to remove insoluble impurities. This separation pathway design helps avoid membrane fouling and flux decline caused by lecithin micelles when the two materials are mixed and then directly ultrafiltered, ensuring a more stable separation process.

[0011] Ultimately, to improve the application performance of the product, this method employs a sequential addition of four modifiers to construct a multilayer hydrophilic structure on the surface of lecithin-peptide composite particles. The process begins by adjusting the pH value to allow the particle surface to carry an appropriate amount of charge, and adding sodium citrate as a charge stabilizer. Next, the addition of sucrose fatty acid esters anchors the hydrophobic surface of the particles with its lipophilic ends, while the hydrophilic ends face the aqueous phase, constructing an initial emulsification interface. Under the action of an ultrasonic field, polyethylene glycol 400 participates in the hydrophilization and dispersion regulation of the particle surface, forming a steric hindrance layer. The final step involves the addition of sodium alginate, which facilitates the formation of a hydration protective structure on the outer layer of the particles. This series of synergistic effects improves the hydrophilic dispersion state of the composite particles, resulting in a final product with good rehydration solubility.

[0012] Preferably, in step S1, the step of obtaining egg yolk powder by freeze-drying after pulsed ultrasonic disruption includes: subjecting the egg yolk homogenate to pulsed ultrasonic disruption, wherein the ultrasonic frequency of the pulsed ultrasonic disruption is 20-40 kHz, the ultrasonic power is 200-500 W, the ultrasonic time is 10-20 min, and a pulse mode with a working time of 2-4 seconds and an interval of 0.5-2 seconds is adopted, and the temperature of the egg yolk homogenate during ultrasonic disruption is 25-30℃; and freeze-drying the egg yolk homogenate after pulsed ultrasonic disruption, wherein the freeze-drying temperature is -55 to -45℃, the vacuum degree is 10-20 Pa, to obtain the egg yolk powder. Simultaneously, fresh crocodile egg yolks, after being mixed with deionized water to form an egg yolk homogenate, can be stored under frozen conditions, thawed to 20-30℃ and stirred until no obvious particles are present before pulsed ultrasonic disruption.

[0013] By employing the above technical solution and using pulsed ultrasound, the intervals allow time for the collapse of acoustic cavitation bubbles and the dissipation of heat from the system. This ensures efficient crushing while precisely controlling the system temperature within the 25-30°C range, where proteins are less prone to denaturation, thus preserving the structural stability of crocodile egg yolk protein. Thawing and stirring before use ensure the homogeneity of the material, guaranteeing the stability and repeatability of the ultrasonic treatment.

[0014] Preferably, in step S2: the step of obtaining defatted residue from the egg yolk powder by supercritical CO2 extraction includes: subjecting the egg yolk powder to supercritical CO2 extraction, wherein the extraction temperature of the supercritical CO2 extraction is 36-49℃, the extraction pressure is 25-35MPa, the CO2 flow rate is 20-50L / h, and the continuous dynamic extraction time is 180-240min, to obtain the defatted residue; the step of adding ethanol to the defatted residue for extraction and centrifugation, using the supernatant as a crude lecithin extract, and de-alcoholizing the precipitated residue under reduced pressure to obtain alcohol-free protein residue. The process includes: adding ethanol to the defatted residue for extraction, wherein the ethanol has a purity of 95% by mass and the amount of ethanol added is 2500-5000 parts by weight relative to 500 parts by weight of the fresh crocodile egg yolk; the extraction temperature is 35-45°C, the rotation speed is 100-200 r / min, and the time is 60-90 min; after extraction, centrifugation is performed, the supernatant is collected as the crude lecithin extract, and the precipitated residue is subjected to vacuum de-alcoholization at 35-45°C to obtain the alcohol-free protein residue.

[0015] By employing the above-mentioned technical solution, supercritical extraction is performed in the near-critical point region, ensuring sufficient density and solubility of the CO2 fluid while avoiding the damage of heat-sensitive substances such as lecithin caused by high temperatures. Subsequent ethanol extraction and vacuum deethanolination are both carried out at a mild temperature of 35–45°C, forming a low-temperature operation chain throughout the entire process, which is beneficial for maintaining the stability of relevant components in the raw materials and intermediate products.

[0016] Preferably, in step S3: the step of adding deionized water to the alcohol-free protein residue, hydrolyzing it with alkaline protease and neutral protease in sequence, modifying it with phospholipase, and centrifuging to obtain a crude polypeptide extract includes: adding 2500-4000 parts by weight of the deionized water to the alcohol-free protein residue relative to 500 parts by weight of the fresh crocodile egg yolk to form a mixed solution; adjusting the pH of the mixed solution to 7.5-8.5, adding 12.5-40 parts by weight of the alkaline protease, and maintaining a constant temperature of 37-45°C. The alkaline hydrolysate is obtained after hydrolysis for 1-2 hours. The pH of the alkaline hydrolysate is adjusted to 7.0-7.5, and 25-80 parts by weight of the neutral protease are added. The hydrolysate is then kept at 37-45°C for 1-2 hours to obtain a neutral hydrolysate. 10-40 parts by weight of the phospholipase are added to the neutral hydrolysate, and the reaction is carried out at 40-50°C for 60 minutes. After the reaction, the temperature is raised to 85-95°C and kept at 85-95°C for 5-15 minutes to inactivate the enzyme. After cooling, the mixture is centrifuged, and the supernatant is collected as the crude polypeptide extract. The step of obtaining ultrafiltration permeate by microfiltration and ultrafiltration of the crude polypeptide extract includes: passing the crude polypeptide extract sequentially through a 150-250 mesh plate and frame filter, a 300-500 mesh paperboard filter, and a 0.1-0.45 μm microfiltration membrane, and then through an ultrafiltration membrane with a molecular weight cutoff of 1000-5000 Da, an operating pressure of 0.1-0.3 MPa, and an operating temperature of 25-30 °C, and collecting the ultrafiltration permeate; the microfiltration step of the crude lecithin extract includes: passing the crude lecithin extract separately through a microfiltration membrane with a pore size of 0.1-0.45 μm.

[0017] By employing the above-mentioned technical solution and using compound enzymatic hydrolysis, the specificity of different proteases for peptide bond cleavage sites is utilized. Through stepwise pH adjustment, the two enzymes act sequentially under their respective optimal conditions, thereby promoting the hydrolysis of crocodile egg yolk protein peptide chains. This improves the degree of hydrolysis and yields the target small molecule peptides. The ultrafiltration membrane's molecular weight cutoff range of 1000–5000 Da allows for precise separation of the target active peptide components, ensuring the purity and bioactivity of the peptides in the final product.

[0018] Preferably, in step S4: relative to 500 parts by weight of the fresh crocodile egg yolk, the amount of sodium citrate added is 12.5-40 parts by weight, the amount of sucrose fatty acid ester added is 25-120 parts by weight, the amount of polyethylene glycol 400 added is 50-200 parts by weight, and the amount of sodium alginate added is 5-20 parts by weight. The step of adjusting the pH value of the lecithin polypeptide mixture includes: controlling the temperature of the lecithin polypeptide mixture to 30-35°C and adjusting the pH value of the lecithin polypeptide mixture to 7.8-8.2; the step of adding sodium citrate and sucrose fatty acid ester and stirring includes: adding sodium citrate and sucrose fatty acid ester to the lecithin polypeptide mixture after pH adjustment, and continuously stirring at a speed of 100-200 r / min; the step of adding polyethylene glycol 400 under ultrasound includes: controlling the ultrasound... The polyethylene glycol 400 was added dropwise at a uniform rate over 10 minutes under an ultrasonic field with an acoustic frequency of 20 kHz and an ultrasonic power of 150 W, followed by continued ultrasonic treatment for 15–25 minutes. The step of adding sodium alginate and stirring to obtain the modified mixture included: maintaining the temperature of the lecithin polypeptide mixture at 30–35 °C and the pH at 7.8–8.2, adding sodium alginate at a uniform rate over 5 minutes, and continuously stirring at 100–200 r / min for 20–30 minutes to obtain the modified mixture. The vacuum concentration was carried out at 40–50 °C and a pressure of -0.09–-0.06 MPa; the freeze-drying was carried out at a freezing temperature of -55–-45 °C and a vacuum degree of 10–20 Pa for 24–36 hours.

[0019] By employing the above technical solution, the pH value is controlled at a weakly alkaline range of 7.8–8.2, which facilitates the dissociation of protein and lecithin surface groups, providing more sites for the subsequent binding of modifiers. Ultrasonic-assisted dropwise addition and subsequent treatment ensure that polyethylene glycol 400 can overcome liquid viscosity resistance, achieving uniform and tight coating and avoiding localized aggregation. Subsequent low-speed stirring allows the sodium alginate molecular chains to slowly and orderly unfold on the outer layer of the particles, forming a uniform and stable final hydration layer. The final low-temperature vacuum concentration and freeze-drying steps continue the protection of heat-sensitive substances throughout the process.

[0020] Preferably, in step S4: the average molecular weight of the polyethylene glycol 400 is 380-420; The monoester content of the sucrose fatty acid ester is greater than or equal to 70% by mass; the sodium alginate is a natural polysaccharide with a weight average molecular weight of 100,000 to 300,000 Da.

[0021] By adopting the above technical solutions, the specifications of the modifier were limited. Sucrose fatty acid esters with high monoester content exhibit stronger emulsifying ability and interfacial affinity; polyethylene glycol 400 with a specific molecular weight can maintain good water solubility while providing sufficient steric hindrance; sodium alginate within a specific molecular weight range can ensure viscosity and film-forming properties while avoiding excessively high system viscosity due to excessively large molecular weight, which would be detrimental to subsequent concentration and drying. These limitations collectively ensure the stability and controllability of the modification effect.

[0022] This invention provides a method for extracting lecithin polypeptides from crocodile egg yolks. It has the following beneficial effects: 1. This invention effectively breaks down the dense granular structure of crocodile egg yolks using pulsed ultrasound, combined with continuous supercritical carbon dioxide extraction, to precisely remove triglycerides and cholesterol. This pretreatment stage solves the problems of low release rate and poor product purity caused by tight lipid binding in traditional processes. It not only avoids the risk of toxic solvent residues but also lays a pure material foundation for the subsequent efficient enrichment of lecithin and the preparation of highly active peptides.

[0023] 2. This invention eliminates residual organic solvents that can inactivate enzymes by introducing a vacuum-controlled dealcoholization process before enzymatic hydrolysis, ensuring a high conversion rate of crocodile egg yolk protein. Simultaneously, a split-stream filtration strategy is employed in the extraction and purification process, allowing the polypeptide hydrolysate and crude lecithin extract to pass through separate filter membranes of corresponding pore sizes before merging. This avoids membrane fouling and pore blockage caused by lipid micellar co-filtration, significantly improving the final yield of the two components.

[0024] 3. This invention modifies the extracted mixture using a multi-component synergistic crosslinking technique, sequentially constructing a hydrophilic network on the particle surface using a dissociation agent and a polymer. This modification step effectively shields the inherent hydrophobic groups in the polypeptides and lecithin derived from crocodile egg yolk, fundamentally overcoming the defects of conventional products that easily aggregate and separate into layers upon entering water. This greatly enhances the rehydration solubility and macroscopic dispersion stability of the finished product, broadening its application range in aqueous nutritional preparations. Attached Figure Description

[0025] Figure 1 The process changes are shown in the diagrams used to verify the extraction and dealcoholization mechanism of this invention; (a) is a cumulative mass monitoring trend diagram of the continuous dynamic extraction process; (b) is a peptide hydrolysis degree monitoring trend diagram of the continuous degradation reaction process. Figure 2 This is a graph showing the flux variation of the ultrafiltration membrane used to verify the multi-scale physical sieving mechanism of this invention. Figure 3 The diagram shows the evolution of microscopic parameters during the hydrophilic modification process of the present invention; wherein, (a) is the evolution trajectory of Zeta potential at each stage of hydrophilic network assembly; and (b) is the evolution trajectory of polydispersity index at each stage of hydrophilic network assembly. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0028] Fresh crocodile egg yolks are taken from healthy, fresh eggs raised in captivity. The egg membrane and impurities are removed, and the contents of the yolk are kept for later use.

[0029] Alkaline protease, CAS number 9014-01-1, enzyme activity limit is 2.4 AU-A / g.

[0030] Neutral protease, CAS number 9068-59-1, with an enzyme activity limit of 1.5 AU / g.

[0031] Phospholipase, CAS number 9001-84-7, enzyme activity is 10000 U / mL.

[0032] Sodium citrate is sodium citrate dihydrate (CAS No. 6132-04-3), which is a food-grade product.

[0033] Polyethylene glycol 400, CAS number 25322-68-3, is a linear polyether polymer formed by the condensation polymerization of ethylene oxide and water. It has an average molecular weight of 380 to 420 Da and a hydroxyl value of 268 to 294 mg KOH / g.

[0034] Sucrose fatty acid ester, CAS number 37318-31-3, is a nonionic surfactant mixture produced by the esterification reaction of sucrose and stearic acid. Its monoester content is greater than or equal to 70% by mass, and its hydrophilic-lipophilic balance value is 15.

[0035] Sodium alginate, CAS No. 9005-38-3, is a linear block copolymer natural polysaccharide composed of β-D-mannuronic acid and α-L-guluronic acid linked by 1,4-glycosidic bonds. Its weight-average molecular weight ranges from 100,000 to 300,000 Da, and the dynamic viscosity of a 1% sodium alginate aqueous solution at 20°C is 200 to 400 mPa·s.

[0036] Example 1: This example provides a method for extracting lecithin polypeptides from crocodile egg yolks, including the following steps: S1. Take 500 parts by weight of fresh crocodile egg yolks, add 500 parts by weight of deionized water, stir to make a homogenate, freeze at -20℃, thaw to 25℃ before use and stir until there are no obvious particles to obtain egg yolk homogenate; place the egg yolk homogenate in an ultrasonic device, set the ultrasonic frequency to 20kHz and the ultrasonic power to 300W, the ultrasonic time to 15min, adopt the pulse mode of working for 3s and intermittent for 1s, turn on the water bath cooling during the period, and control the temperature of the egg yolk homogenate to 28℃. After the ultrasonic treatment, place the broken egg yolk homogenate in a freeze dryer and dehydrate it under the conditions of freezing temperature -50℃ and vacuum degree 15Pa to obtain egg yolk powder.

[0037] S2. Place the dried egg yolk powder into a supercritical extraction vessel, introduce CO2 gas, set the extraction temperature to 45℃ and the extraction pressure to 32MPa, and the CO2 flow rate to 30L / h, and perform continuous dynamic extraction for 180min. After extraction, separate and remove neutral lipids under reduced pressure, and collect the defatted residue in the extraction vessel. Add 4000 parts by weight of 95% ethanol to the defatted residue, and stir and extract in a 40℃ constant temperature water bath at 150r / min for 75min. Centrifuge at 5000r / min for 15min, and separate and collect the supernatant as the crude lecithin extract. Place the precipitate obtained after centrifugation in a vacuum drying oven and evaporate the residual ethanol under reduced pressure at 40℃ to obtain alcohol-free protein residue.

[0038] S3. Add 3000 parts by weight of deionized water to the alcohol-free protein residue and stir at 150 r / min to disperse it evenly. Adjust the pH of the mixture of alcohol-free protein residue and deionized water to 8.1 using sodium hydroxide solution. Add 20 parts by weight of alkaline protease and hydrolyze at 40℃ for 1 hour to obtain an alkaline hydrolysate. Then adjust the pH of the alkaline hydrolysate to 7.2 using sodium hydroxide solution, add 40 parts by weight of neutral protease, and continue hydrolyzing at 40℃ for 1 hour to obtain a neutral hydrolysate. Finally, add 10 parts by weight of phospholipase to the neutral hydrolysate and react at 45℃ for 6 hours. 0 min; after the reaction, heat to 90℃ and incubate for 10 min to inactivate the enzyme. After cooling, centrifuge at 6000 r / min for 20 min and collect the supernatant as the crude peptide extract. Pass the crude peptide extract sequentially through a 200-mesh plate and frame, a 400-mesh paperboard and a 0.22 μm microfiltration membrane, and then through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, an operating pressure of 0.2 MPa and an operating temperature of 28℃. Collect the ultrafiltration permeate. At the same time, pass the crude lecithin extract separately through a 0.22 μm microfiltration membrane to obtain lecithin microfiltrate. Mix the ultrafiltration permeate and lecithin microfiltrate to obtain a lecithin peptide mixture.

[0039] S4. The lecithin peptide mixture was kept at 32°C. The pH of the lecithin peptide mixture was adjusted and locked to 8.0 using sodium hydroxide solution. 15 parts by weight of sodium citrate and 60 parts by weight of sucrose fatty acid ester were added sequentially, and the mixture was continuously stirred at 150 r / min for dissociation. Then, the ultrasonic generator was turned on and the ultrasonic frequency was set to 20 kHz and the power to 150 W. 90 parts by weight of polyethylene glycol 400 were added dropwise to the lecithin peptide mixture at a uniform rate over 10 min under ultrasonic field, and the mixture was ultrasonically treated for 20 min. The temperature and pH were kept stable at 32°C and 8.0. 12 parts by weight of sodium alginate were added at a uniform rate over 5 min, and the mixture was continuously stirred at 150 r / min for 25 min to obtain a modified mixture. The modified mixture was concentrated under reduced pressure at 45°C and -0.08 MPa, and then placed in a freeze dryer and freeze-dried at -50°C and 15 Pa for 30 h to obtain the crocodile egg yolk lecithin peptide product.

[0040] Example 2: This example provides a method for extracting lecithin polypeptides from crocodile egg yolks, including the following steps: S1. Take 500 parts by weight of fresh crocodile egg yolks, add 500 parts by weight of deionized water, stir to make a homogenate, freeze at -20℃, and before use, thaw to 20℃ and stir until there are no obvious particles to obtain egg yolk homogenate; place the egg yolk homogenate in an ultrasonic device, set the ultrasonic frequency to 30kHz and the ultrasonic power to 200W, the ultrasonic time to 10min, adopt the pulse mode of working for 2s and intermittent for 0.5s, turn on the water bath cooling during the period, and control the temperature of the egg yolk homogenate to 25℃. After the ultrasonic treatment, place the broken egg yolk homogenate in a freeze dryer and dehydrate it under the conditions of freezing temperature -45℃ and vacuum degree 10Pa to obtain egg yolk powder.

[0041] S2. Place the dried egg yolk powder into a supercritical extraction vessel, introduce CO2 gas, set the extraction temperature to 36℃ and the extraction pressure to 25MPa, and the CO2 flow rate to 20L / h, and perform continuous dynamic extraction for 200min. After extraction, separate and remove neutral lipids under reduced pressure, and collect the defatted residue in the extraction vessel. Add 2500 parts by weight of 95% ethanol to the defatted residue, and stir and extract in a 35℃ constant temperature water bath at 100r / min for 60min. Centrifuge at 4000r / min for 10min, and separate and collect the supernatant as the crude lecithin extract. Place the precipitate obtained after centrifugation in a vacuum drying oven and evaporate the residual ethanol under reduced pressure at 35℃ to obtain alcohol-free protein residue.

[0042] S3. Add 2500 parts by weight of deionized water to the alcohol-free protein residue and stir at 100 r / min to disperse it evenly. Adjust the pH of the mixture of alcohol-free protein residue and deionized water to 7.5 using sodium hydroxide solution. Add 12.5 parts by weight of alkaline protease and hydrolyze at 37℃ for 1.5 h to obtain an alkaline hydrolysate. Then adjust the pH of the alkaline hydrolysate to 7.0 using sodium hydroxide solution. Add 25 parts by weight of neutral protease and continue hydrolyzing at 37℃ for 1.5 h to obtain a neutral hydrolysate. Finally, add 12.5 parts by weight of phospholipase to the neutral hydrolysate and hydrolyze at 40℃. The reaction was carried out at a warm temperature for 60 min. After the reaction, the temperature was raised to 85℃ and kept at 85℃ for 15 min to inactivate the enzyme. After cooling, the mixture was centrifuged at 5000 r / min for 15 min, and the supernatant was collected as the crude peptide extract. The crude peptide extract was then passed sequentially through a 150-mesh plate and frame filter, a 300-mesh paperboard filter, and a 0.45 μm microfiltration membrane. It was then passed through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, an operating pressure of 0.1 MPa, and an operating temperature of 25℃. The ultrafiltration permeate was collected. At the same time, the crude lecithin extract was passed separately through a 0.45 μm microfiltration membrane to obtain the lecithin microfiltrate. The ultrafiltration permeate and the lecithin microfiltrate were mixed to obtain the lecithin peptide mixture.

[0043] S4. The lecithin peptide mixture was kept at 30°C. The pH of the lecithin peptide mixture was adjusted and locked at 7.8 using sodium hydroxide solution. 12.5 parts by weight of sodium citrate and 25 parts by weight of sucrose fatty acid ester were added sequentially, and the mixture was continuously stirred at 100 r / min for dissociation. Then, the ultrasonic generator was turned on and the ultrasonic frequency was set to 20 kHz and the power to 150 W. 50 parts by weight of polyethylene glycol 400 were added dropwise to the lecithin peptide mixture at a uniform rate over 10 min under ultrasonic field, and the mixture was ultrasonically treated for 15 min. The temperature and pH were kept stable at 30°C and 7.8. 5 parts by weight of sodium alginate were added at a uniform rate over 5 min, and the mixture was continuously stirred at 100 r / min for 20 min to obtain a modified mixture. The modified mixture was concentrated under reduced pressure at 40°C and -0.06 MPa, and then placed in a freeze dryer and freeze-dried at -45°C and 10 Pa for 24 h to obtain the crocodile egg yolk lecithin peptide product.

[0044] Example 3: This example provides a method for extracting lecithin polypeptides from crocodile egg yolks, including the following steps: S1. Take 500 parts by weight of fresh crocodile egg yolks, add 500 parts by weight of deionized water, stir to make a homogenate, freeze at -20℃, thaw to 30℃ before use and stir until there are no obvious particles to obtain egg yolk homogenate; place the egg yolk homogenate in an ultrasonic device, set the ultrasonic frequency to 40kHz and the ultrasonic power to 500W, the ultrasonic time to 20min, adopt the pulse mode of working for 4s and intermittent for 2s, turn on the water bath cooling during the period, and control the temperature of the egg yolk homogenate to 30℃. After the ultrasonic treatment, place the broken egg yolk homogenate in a freeze dryer and dehydrate it under the conditions of freezing temperature -55℃ and vacuum degree 20Pa to obtain egg yolk powder.

[0045] S2. Place the dried egg yolk powder into a supercritical extraction vessel, introduce CO2 gas, set the extraction temperature to 49℃ and the extraction pressure to 35MPa, and the CO2 flow rate to 50L / h, and perform continuous dynamic extraction for 240min. After extraction, separate and remove neutral lipids under reduced pressure, and collect the defatted residue in the extraction vessel. Add 5000 parts by weight of 95% pure ethanol to the defatted residue, and stir and extract in a constant temperature water bath at 45℃ at 200r / min for 90min. Centrifuge at 6000r / min for 20min, and separate and collect the supernatant as crude lecithin extract. Place the precipitate obtained after centrifugation in a vacuum drying oven and evaporate the residual ethanol under reduced pressure at 45℃ to obtain alcohol-free protein residue.

[0046] S3. Add 4000 parts by weight of deionized water to the alcohol-free protein residue and stir at 200 rpm to disperse it evenly. Adjust the pH of the mixture of alcohol-free protein residue and deionized water to 8.5 using sodium hydroxide solution. Add 40 parts by weight of alkaline protease and hydrolyze at 45°C for 2 hours to obtain an alkaline hydrolysate. Then adjust the pH of the alkaline hydrolysate to 7.5 using sodium hydroxide solution, add 80 parts by weight of neutral protease, and continue hydrolyzing at 45°C for 2 hours to obtain a neutral hydrolysate. Finally, add 40 parts by weight of phospholipase to the neutral hydrolysate and react at 50°C. The reaction was carried out for 60 min. After the reaction, the temperature was raised to 95℃ and kept at that temperature for 5 min to inactivate the enzyme. After cooling, the mixture was centrifuged at 7000 r / min for 25 min, and the supernatant was collected as the crude peptide extract. The crude peptide extract was then passed sequentially through a 250-mesh plate and frame filter, a 500-mesh paperboard filter, and a 0.1 μm microfiltration membrane. It was then passed through an ultrafiltration membrane with a molecular weight cutoff of 5000 Da, an operating pressure of 0.3 MPa, and an operating temperature of 30℃. The ultrafiltration permeate was collected. At the same time, the crude lecithin extract was passed separately through a 0.1 μm microfiltration membrane to obtain the lecithin microfiltrate. The ultrafiltration permeate and the lecithin microfiltrate were mixed to obtain the lecithin peptide mixture.

[0047] S4. The lecithin peptide mixture was heated to 35°C, and the pH of the lecithin peptide mixture was adjusted and locked to 8.2 using sodium hydroxide solution. 40 parts by weight of sodium citrate and 120 parts by weight of sucrose fatty acid ester were added sequentially, and the mixture was continuously stirred at 200 r / min for dissociation. Then, the ultrasonic generator was turned on and the ultrasonic frequency was set to 20 kHz and the power to 150 W. Under the ultrasonic field, 200 parts by weight of polyethylene glycol 400 were added dropwise to the lecithin peptide mixture at a uniform rate over 10 min, and the mixture was ultrasonically treated for 25 min. The temperature was kept stable at 35°C and the pH was 8.2. 20 parts by weight of sodium alginate were added at a uniform rate over 5 min, and the mixture was continuously stirred at 200 r / min for 30 min to obtain a modified mixture. The modified mixture was concentrated under reduced pressure at 50°C and -0.09 MPa, and then placed in a freeze dryer and freeze-dried at -55°C and 20 Pa for 36 h to obtain the crocodile egg yolk lecithin peptide product.

[0048] Comparative Example 1: Compared with Example 1, the difference is that the modification operation in step S4 was not performed. Specifically, the lecithin peptide mixture obtained in step S3 was concentrated under reduced pressure at 45°C and -0.08 MPa without any pH adjustment or addition of modifiers, and then freeze-dried in a freeze dryer at -50°C and 15 Pa for 30 hours. The remaining steps and parameters were the same as in Example 1.

[0049] Comparative Example 2: Compared with Example 1, the difference is that the pulsed ultrasonic disruption process in step S1 was removed. Specifically, fresh crocodile egg yolks were mixed with deionized water, thawed to 25°C, and stirred until no obvious particles were observed. The resulting egg yolk homogenate was then directly placed in a freeze dryer for dehydration at a freezing temperature of -50°C and a vacuum degree of 15Pa to obtain egg yolk powder. That is, ultrasonic treatment was not performed in a pulse mode with a set frequency of 20kHz, a power of 300W, a working time of 3 seconds, and a 1-second interval. All other steps and parameters were the same as in Example 1.

[0050] Comparative Example 3: Compared with Example 1, the difference is that the supercritical CO2 continuous dynamic extraction process in step S2 was removed. Specifically, the egg yolk powder obtained in step S1 was directly added to 4000 parts by weight of 95% pure ethanol and extracted in a constant temperature water bath at 40°C; that is, it was not placed in a supercritical extraction vessel to introduce CO2 gas for extraction, separation, and removal of lipids (neutral lipids / cholesterol, etc.). The remaining steps and parameters were the same as in Example 1.

[0051] Comparative Example 4: Compared with Example 1, the difference is that all enzymatic hydrolysis processes in step S3 were removed. Specifically, deionized water was added to the alcohol-free protein residue obtained in step S2 and stirred until evenly dispersed. Alkaline protease, neutral protease, and phospholipase were not added for isothermal hydrolysis and enzyme inactivation. The supernatant was collected by centrifugation at 6000 r / min for 20 min and mixed with lecithin microfiltrate according to the subsequent membrane separation and purification steps of Example 1. All other steps and parameters were the same as in Example 1.

[0052] Comparative Example 5: Compared with Example 1, the difference is that no phospholipase was added for modification during the enzymatic hydrolysis stage in step S3. Specifically, in step S3, 40 parts by weight of neutral protease were added and hydrolyzed at 40°C for 1 hour, followed by direct heating to 90°C and incubation for 10 minutes to inactivate the enzyme; thus, the step of adding 10 parts by weight of phospholipase at the end and reacting at 45°C for 60 minutes was removed. The remaining steps and parameters were the same as in Example 1.

[0053] Comparative Example 6: Compared with Example 1, the difference lies in the membrane separation and purification operation of the crude peptide extract and crude lecithin extract in step S3. Specifically, after collecting the crude peptide extract by centrifugation at 6000 r / min for 20 min in step S3, the crude lecithin extract collected in step S2 is directly mixed with the crude peptide extract to obtain a crude mixture; that is, the operation of filtering the crude peptide extract and crude lecithin extract separately and then mixing the ultrafiltration permeate with the lecithin microfiltration solution in Example 1 is removed. Instead, the above crude mixture is passed sequentially through a 200-mesh plate and frame filter, a 400-mesh paperboard filter, and a 0.22 μm microfiltration membrane, and then through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, an operating pressure of 0.2 MPa, and an operating temperature of 28 °C. The ultrafiltration permeate is collected and directly used as the lecithin-peptide mixture in step S4. The remaining steps and parameters are the same as in Example 1.

[0054] Test Example 1:

[0055] Experimental steps: Equal amounts of the egg yolk powder prepared in Example 1 and the unultrasonicated egg yolk powder prepared in Comparative Example 2 were weighed. Both samples were placed into identical supercritical extraction vessels, and CO2 gas was introduced. The extraction temperature was set to 45°C, the extraction pressure to 32 MPa, and the CO2 flow rate to a constant 30 L / h for continuous dynamic extraction. The monitoring time was recorded starting from the beginning of CO2 introduction. At different time points, the precipitated neutral lipids were discharged through a pressure-reducing valve and weighed using an analytical balance. The weights of all neutral lipids collected before each time point were summed to obtain the cumulative extracted lipid mass of the powder from Example 1 and the cumulative extracted lipid mass of the unultrasonicated powder from Comparative Example 2 at different monitoring times.

[0056] Equal amounts of the alcohol-free protein residue prepared in step S2 of Example 1 and the precipitate containing residual ethanol retained directly after centrifugation in step S2 without vacuum drying were taken. These two residues were placed in separate reaction vessels, deionized water was added, and the mixture was stirred to disperse it evenly. A suitable concentration of NaOH solution was added dropwise to adjust the pH of the mixture to 8.1. The reaction vessels were then placed in a 40°C constant-temperature water bath. An equal amount of alkaline protease from the same batch was added to the liquid surface. An automatic potentiometric titrator was used to maintain a constant pH of 8.1. During the reaction, the instrument automatically added NaOH standard solution of a known concentration according to the pH change. The monitoring time was recorded from the moment the protease was added. The cumulative volume of NaOH solution consumed at different time points was read. The degree of peptide hydrolysis of the alcohol-free residue and the ethanol-containing residue in Example 1 were calculated based on the conversion formula between the consumed alkaline solution volume and the total nitrogen content, thus obtaining the degree of peptide hydrolysis at different monitoring times.

[0057] Experimental results (see Table 1 and Table 2): Table 1: Comparison of Cumulative Extracted Lipid Mass in Different Powder Forms

[0058] Table 2: Comparison of peptide hydrolysis degree in different residue states

[0059] Test conclusion: According to Table 1, Table 2 and Figure 1As shown, the egg yolk powder of Example 1, after 180 min of extraction, yielded a cumulative lipid extraction mass of 161.42 g, which remained relatively stable. In contrast, the equivalent mass of untreated powder from Comparative Example 2, after 300 min of extraction, yielded only 126.51 g of lipid extraction. These results indicate that the untreated egg yolk powder retained a dense particulate structure, increasing the mass transfer resistance of supercritical CO2 fluid into the internal pores, resulting in a lower extraction rate of neutral lipids. Example 1, through pulsed ultrasonic treatment, disrupted the egg yolk particulate structure, promoting full lipid exposure and thus improving the extraction efficiency of supercritical fat removal and separation.

[0060] In Example 1, the degree of hydrolysis of the alcohol-free protein residue steadily increased over time after the addition of protease, reaching 18.52% at 60 min. In contrast, the precipitated residue containing residual ethanol showed a slow enzymatic hydrolysis reaction, with a degree of hydrolysis of only 4.21% at 60 min. This data comparison indicates that residual ethanol in the residue, upon entering the aqueous phase, causes conformational changes in the protease and leads to a decrease in the activity of some enzymes, thereby reducing the reaction efficiency between the enzyme and the substrate. Performing vacuum evaporation and alcohol removal before the enzymatic hydrolysis process removes residual organic solvents that easily inactivate biological enzymes, providing a suitable aqueous phase environment for the subsequent proteolytic hydrolysis process and ensuring the high conversion rate of the peptide hydrolysis.

[0061] Test Example 2:

[0062] Experimental steps: 1000 mL of the crude peptide extract collected by centrifugation in step S3 of Example 1 and the crude mixture obtained by pre-mixing the crude lecithin extract and crude peptide extract in Comparative Example 6 were measured separately. Both test solutions were pumped into ultrafiltration membrane modules with the same effective membrane area and a molecular weight cutoff of 1000 Da. The separation equipment was turned on, and the transmembrane pressure difference was set to 0.2 MPa, and the operating temperature to 28°C. The moment the feed solution began to enter the ultrafiltration membrane module was taken as the starting point for timing. At each set operating time point, the cumulative volume of the permeate was collected and recorded using a graduated cylinder. Based on the recorded permeate volume, the effective membrane area of ​​the membrane module, and the corresponding sampling time interval, calculations were performed to obtain the ultrafiltration membrane flux of the crude peptide extract of Example 1 and the crude mixture of Comparative Example 6 at different operating time points.

[0063] After all the feed solutions have completed the ultrafiltration separation process, the waste liquids discharged from the ultrafiltration membrane retentate side of Example 1 and Comparative Example 6 are collected respectively. The two sets of waste liquids are placed in a vacuum drying oven and dried continuously at a temperature of 60°C and a vacuum degree of 0.08 MPa until the weight no longer changes. Then, they are taken out and accurately weighed using an analytical balance to obtain the dry matter weight of the retentate waste liquid in the separation section of Example 1 and the dry matter weight of the retentate waste liquid in the separation section of Comparative Example 6.

[0064] Experimental results (see Tables 3 and 4): Table 3: Comparison of Ultrafiltration Membrane Flux under Different Feed Conditions

[0065] Table 4: Comparison of Dry Weight of Retained Waste Liquid in Different Separation Stages

[0066] Test conclusion: According to Table 3, Table 4 and Figure 2 As shown, the crude peptide extract of Example 1 maintained good water permeability during ultrafiltration separation, with the ultrafiltration membrane flux remaining at 41.3 L / m³ after 30 min. 2 The dry matter content of the waste liquid retained was only 14.2 g. In contrast, the coarse mixture of Comparative Example 6 exhibited severe membrane fouling under the same conditions, with the flux dropping to 8.2 L / m³ after only 15 minutes of ultrafiltration. 2 ·h, ultimately the amount of dry matter retained in the waste liquid increased to 61.5g.

[0067] The difference arises because lecithin readily aggregates in aqueous systems to form large liposome micelles. If peptides and lecithin are pre-mixed and co-filtered, these large lipid micelles rapidly adhere to the surface of the 1000 Da ultrafiltration membrane, clogging the pores and stacking to form a water-blocking gel layer, leading to a rapid decrease in filtration flux. Simultaneously, this surface gel layer intercepts smaller peptide molecules that would otherwise pass through the pores, causing the active ingredients to be lost with the waste liquid. Example 1, by establishing a split purification channel, allows extracts of different molecular sizes to be separated by membrane separation. This process avoids lipid micelle contamination and pore retention of the ultrafiltration membrane, maintaining a high membrane flux and thus ensuring the final extraction yield of peptides and lecithin.

[0068] Test Example 3:

[0069] Experimental steps: Lecithin polypeptide mixtures obtained in step S3 of Example 1, lecithin polypeptide mixtures obtained by adding sodium citrate and sucrose fatty acid esters and continuously stirring and dissociating them at 150 r / min in step S4 of Example 1, and lecithin polypeptide mixtures obtained by adding polyethylene glycol 400 and ultrasonic treatment (ultrasonic frequency 20 kHz and power 150 W) in step S4 of Example 1 were extracted separately. These three liquid samples at different assembly stages were injected into the standard test cells of a Zeta potentiometer and a dynamic light scattering instrument, respectively, and detected under constant temperature conditions of 25℃. The Zeta potentiometer calculates the Zeta potential of the corresponding liquid sample by measuring the electrophoretic mobility of suspended particles under the action of an electric field; the dynamic light scattering instrument analyzes the polydispersity index of the corresponding liquid sample by capturing the intensity fluctuations of scattered light caused by the Brownian motion of particles.

[0070] Weigh out 2.0g of the alligator egg yolk lecithin peptide product prepared in Example 1 and the unmodified alligator egg yolk lecithin peptide product from Comparative Example 1. Sprinkle both powders evenly into two identical 250mL beakers containing 100mL of deionized water from the same height (5cm) above the liquid surface. Use a stopwatch to observe and time the process, starting at the moment the powder particles contact the water surface and ending when the powder is completely submerged and sinks to the bottom of the beaker. Record the total time taken to obtain the wetting and sinking time for the product of Example 1 and Comparative Example 1.

[0071] Experimental results (see Tables 5 and 6): Table 5: Monitoring Table of Microscopic Interface Parameters at Each Stage of Assembly

[0072] Table 6: Comparison of wetting and sinking times for different powder states

[0073] Test conclusion: According to Table 5, Table 6 and Figure 3 The data shows that after adding sodium citrate and sucrose fatty acid ester, the Zeta potential of the mixture in step S3 of Example 1 decreased from the initial -12.43 mV to -38.62 mV. This indicates that the surface charge state of the particles changed, the electrostatic repulsion between particles increased, and this, together with the interfacial regulation effect of sucrose fatty acid ester, affected the dispersion stability of the colloidal particles. After introducing polyethylene glycol 400, the polydispersity index of the liquid sample decreased from 0.45 to 0.18, reflecting that polyethylene glycol 400 participated in the hydrophilization and dispersion regulation of the particle interface under ultrasonic treatment, reducing the dispersion of the particle size distribution in the system.

[0074] The crocodile egg yolk lecithin polypeptide product of Example 1 took only 8.2 seconds to wet and sink after contacting the water surface; while the product of Comparative Example 1, which had not undergone hydrophilic modification, formed hydrophobic agglomerates and floated on the liquid surface after contacting the aqueous phase, with a wettability and sinking time of 124.6 seconds. This comparison confirms that Example 1, through the compound crosslinking of sodium citrate, sucrose fatty acid ester, polyethylene glycol 400, and sodium alginate, constructed a hydrophilic network structure on the surface of the powder particles, which improved the wetting and dispersion process of the powder when in contact with water, solved the problem of easy agglomeration of the unmodified product in water, and improved the dispersion and solubility of the final material.

[0075] Test Example 4:

[0076] Experimental steps: The crocodile egg yolk lecithin polypeptide products finally prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were selected as the powder samples to be tested.

[0077] Accurately weigh 1.0g of each group of powder samples to be tested, and determine the nitrogen content using the Kjeldahl method. Multiply the result by a conversion factor of 6.12 to obtain the protein content. Accurately weigh 0.5g of each group of powder samples to be tested, dissolve them, and dilute to 50mL to prepare a test solution. Quantitatively analyze each sample using high-performance liquid chromatography (HPLC), and calculate the peptide, lecithin, and cholesterol contents based on the standard curve. Simultaneously, accurately weigh 2.0g of each group of powder samples to be tested, and determine the total lipid content using the conventional Soxhlet extraction gravimetric method. Divide the previously determined lecithin content by the total lipid content to obtain the percentage of phospholipids in the total lipids.

[0078] In the solubility test, under 25℃ conditions, each group of test powder samples was slowly added in batches to beakers containing 100mL of deionized water. The mixture was stirred continuously at 200r / min until visible undissolved particles appeared and dissolution ceased. The total mass of the completely dissolved test powder samples was recorded, and the solubility of each product in water was calculated accordingly. 50mL of each group of liquid samples that had reached dissolution equilibrium was transferred and placed in identical 100mL transparent glass colorimetric tubes. The initial phase distribution was directly observed to assess dispersibility. The samples were then allowed to stand at room temperature for 15 minutes and 30 minutes, respectively, and the presence of turbidity, precipitation, or stratification was recorded.

[0079] Experimental results (see Table 7): Table 7: Comparison of Physicochemical Properties and Macroscopic Stability of Different Powder Products

[0080] Test conclusion: As shown in Table 7, the finished products of Examples 1 to 3, while maintaining low cholesterol (all below 0.75 g / 100 g) and high content of active ingredients, exhibit excellent water solubility and static stability. This high purity directly benefits from the impurity removal methods at the extraction source. Comparing the differences in the pre-processing steps, it can be seen that Comparative Example 2, which lacks pulsed ultrasonic cell disruption, retains the dense structure of the egg yolk particles, increasing the mass transfer resistance during extraction and enzymatic hydrolysis, thus reducing the content of peptides and lecithin. If the supercritical carbon dioxide impurity removal process is omitted, as shown in Comparative Example 3, it will result in a large amount of residual neutral lipids, with a cholesterol content as high as 8.97 g / 100 g in the product, reducing the proportion of phospholipids in the total lipids. This confirms the necessity of the combined impurity removal process in the early stage of this invention for improving product purity.

[0081] After establishing a high-purity material base, the hydrolysis and separation purification methods significantly impacted the physicochemical configuration of the product. Comparative Example 4, lacking complex protease degradation, resulted in incomplete protein hydrolysis, a significant decrease in peptide content, and noticeable aqueous sedimentation. Comparative Example 5, without phospholipase modification, failed to further regulate the interfacial compatibility between lecithin and the peptide system, leading to slight stratification of the material during settling. Furthermore, the co-filtration of the crude extract used in Comparative Example 6 resulted in rapid pore blockage of the membrane by large-sized lipid micelles, intercepting a large amount of active substances and reducing the content of effective components. A segmented complex hydrolysis combined with a split-flow membrane purification process is crucial for avoiding membrane fouling while maintaining high yields for both components.

[0082] The differences in final application characteristics are mainly reflected in the macroscopic hydration capacity of the powder. In Comparative Example 1, due to the lack of subsequent hydrophilic modification, the hydrophobic groups inside the finished product caused significant physical aggregation and phase separation, resulting in a solubility reduction to 22.84 g / 100 mL. In the Example, the composite network structure synergistically constructed from sodium citrate, sucrose fatty acid ester, polyethylene glycol 400, and sodium alginate effectively altered the natural wetting properties of the particle surface, improving the problem of easy agglomeration and stratification of the unmodified product upon entering water, thus enabling the product to exhibit good dispersion and solubility.

[0083] Test Example 5:

[0084] Experimental steps: Forty healthy SPF-grade male SD rats, weighing between 200 and 250 g, were selected. The animals were placed in a constant temperature and humidity environment (22±2℃, 50%±10% relative humidity, 12h / 12h light / dark cycle) for basic acclimatization. Subsequently, eight rats were randomly selected as the blank control group (Group A) and continuously fed a standard basal diet. The remaining 32 rats were fed a high-fat diet to establish a hyperlipidemia model. After successful model establishment, the rats fed the high-fat diet were randomly divided into four groups: a high-fat model group (Group B), a low-dose group (Group C, prepared at a dosage of 5 mg / kg body weight), a medium-dose group (Group D, prepared at a dosage of 10 mg / kg body weight), and a high-dose group (Group E, prepared at a dosage of 15 mg / kg body weight).

[0085] In the intervention group, rats were administered gavage at a fixed time each day according to a set dosage. The gavage substance was an aqueous solution prepared from the crocodile egg yolk lecithin polypeptide product obtained in Example 1, with a uniform administration volume of 10 mL / kg body weight. Simultaneously, the blank control group and the high-fat model group were administered an equal volume of physiological saline daily via gavage. This continuous intervention period lasted for 4 weeks. After the intervention period, all rats were fasted for 12 hours. Under anesthesia, venous blood samples were individually collected from each rat. After standing, the samples were centrifuged at 3000 r / min for 10 minutes at 4°C to separate serum samples. The biochemical concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum of each rat were quantitatively detected using a fully automated biochemical analyzer. Statistical calculations were performed on the test data of the 8 rats in each group, and the average value of the data for each group was calculated.

[0086] Experimental results (see Table 8): Table 8: Comparison of serum lipid levels in rats of different groups (mean values ​​of each group, n=8)

[0087] Test conclusion: The biochemical data in Table 8 show that the concentrations of TC, TG, and LDL-C in the high-lipidemia model group were significantly higher than those in the blank control group, and the HDL-C level decreased, proving that the hyperlipidemia model was successfully established. After intervention with the product from Example 1, the abnormal blood lipid indicators in each group of rats showed a dose-related regression trend. Among them, the TC and LDL-C in the high-dose group decreased to 1.65 mmol / L and 0.73 mmol / L, respectively, which were close to the levels of the blank control group.

[0088] In vivo experiments showed that the obtained extract had a regulatory effect on high-fat diet-induced dyslipidemia in rats. This effect was related to the retention of lecithin, peptides, and other components in the finished product and the improvement of their aqueous dispersibility. Supercritical fluid impurity removal removed triglycerides and free cholesterol from the raw material, and split membrane purification helped reduce the loss of lecithin and peptides during the separation process. Combined with supercritical fluid impurity removal, the final extract maintained a low level of impurities.

[0089] Based on this high-purity material, the hydrophilic interpenetrating network constructed on the surface of the finished powder improves the wetting properties of the natural lipopeptide complex, which is beneficial to improving the wetting and dispersion state of the finished product in an aqueous environment, and provides conditions for its dispersion and release after oral ingestion.

[0090] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting lecithin polypeptides from crocodile egg yolks, characterized in that, Includes the following steps: S1. Fresh crocodile egg yolks are mixed with deionized water to make egg yolk homogenate, which is then broken by pulsed ultrasound and freeze-dried to obtain egg yolk powder. S2. The egg yolk powder is extracted with supercritical CO2 to obtain defatted residue; the defatted residue is extracted with ethanol and centrifuged, the supernatant is used as crude lecithin extract, and the precipitated residue is de-alcoholized under reduced pressure to obtain alcohol-free protein residue. S3. The alcohol-free protein residue is added to deionized water, and then hydrolyzed with alkaline protease and neutral protease in sequence. Phospholipase is added for modification, and the enzyme is inactivated by centrifugation to obtain a crude polypeptide extract. The crude polypeptide extract is subjected to microfiltration and ultrafiltration to obtain an ultrafiltration permeate, which is mixed with the microfiltration crude lecithin extract to obtain a lecithin polypeptide mixture. S4. Adjust the pH value of the lecithin polypeptide mixture, add sodium citrate and sucrose fatty acid ester and stir, add polyethylene glycol 400 under ultrasound, add sodium alginate and stir to obtain the modified mixture; The modified mixture was concentrated under reduced pressure and freeze-dried to obtain the finished product; In step S1: The step of obtaining egg yolk powder by freeze-drying after pulsed ultrasonic disruption includes: subjecting the egg yolk homogenate to pulsed ultrasonic disruption, wherein the ultrasonic frequency of the pulsed ultrasonic disruption is 20-40 kHz, the ultrasonic power is 200-500 W, the ultrasonic time is 10-20 min, and a pulse mode with a working time of 2-4 seconds and an interval of 0.5-2 seconds is adopted, and the temperature of the egg yolk homogenate during ultrasonic disruption is 25-30℃; and freeze-drying the egg yolk homogenate after pulsed ultrasonic disruption, wherein the freeze-drying temperature is -55 to -45℃, the vacuum degree is 10-20 Pa, to obtain the egg yolk powder. In step S2: The step of obtaining defatted residue by supercritical CO2 extraction of egg yolk powder includes: supercritical CO2 extraction of egg yolk powder, wherein the extraction temperature of supercritical CO2 extraction is 36-49℃, the extraction pressure is 25-35MPa, the CO2 flow rate is 20-50L / h, and the continuous dynamic extraction time is 180-240min, thereby obtaining the defatted residue; The steps of extracting the defatted residue with ethanol and centrifuging, using the supernatant as a crude lecithin extract, and then subjecting the precipitated residue to vacuum deethanolation to obtain alcohol-free protein residue include: adding ethanol to the defatted residue for extraction, wherein the purity of the ethanol is 95% by mass, and the amount of ethanol added is 2500-5000 parts by weight relative to 500 parts by weight of the fresh alligator egg yolk; the extraction temperature is 35-45°C, the rotation speed is 100-200 r / min, and the time is 60-90 min; after the extraction is completed, centrifuging is performed, the supernatant is collected as the crude lecithin extract, and the precipitated residue is subjected to vacuum deethanolation at 35-45°C to obtain the alcohol-free protein residue; In step S3: The steps of adding deionized water to the alcohol-free protein residue, hydrolyzing it sequentially with alkaline protease and neutral protease, modifying it with phospholipase, and centrifuging to obtain a crude polypeptide extract include: adding 2500-4000 parts by weight of the deionized water to the alcohol-free protein residue relative to 500 parts by weight of the fresh crocodile egg yolk to form a mixture; adjusting the pH of the mixture to 7.5-8.5; adding 12.5-40 parts by weight of the alkaline protease; and hydrolyzing at a constant temperature of 37-45°C for 1-2 hours. An alkaline hydrolysate was obtained; the pH of the alkaline hydrolysate was adjusted to 7.0–7.5, and 25–80 parts by weight of the neutral protease were added. The hydrolysate was continued at 37–45°C for 1–2 hours to obtain a neutral hydrolysate; 10–40 parts by weight of the phospholipase were added to the neutral hydrolysate, and the reaction was carried out at 40–50°C for 60 minutes; after the reaction, the temperature was raised to 85–95°C and kept at 85–95°C for 5–15 minutes to inactivate the enzyme. After cooling, the mixture was centrifuged, and the supernatant was collected as the crude polypeptide extract. In step S4: Relative to 500 parts by weight of the fresh crocodile egg yolk, the amount of sodium citrate added is 12.5 to 40 parts by weight, the amount of sucrose fatty acid ester added is 25 to 120 parts by weight, the amount of polyethylene glycol 400 added is 50 to 200 parts by weight, and the amount of sodium alginate added is 5 to 20 parts by weight.

2. The method for extracting crocodile egg yolk lecithin polypeptides according to claim 1, characterized in that, In step S1: The steps for preparing egg yolk homogenate by adding deionized water to fresh crocodile egg yolk include: after preparing egg yolk homogenate by adding deionized water to fresh crocodile egg yolk, storing it under frozen conditions, thawing it to 20-30°C and stirring it until there are no obvious particles before performing pulsed ultrasonic disruption.

3. The method for extracting crocodile egg yolk lecithin polypeptides according to claim 1, characterized in that, In step S3: The step of obtaining ultrafiltration permeate by microfiltration and ultrafiltration of the crude polypeptide extract includes: passing the crude polypeptide extract sequentially through a 150-250 mesh plate and frame membrane, a 300-500 mesh paperboard membrane, and a 0.1-0.45 μm microfiltration membrane, and then through an ultrafiltration membrane with a molecular weight cutoff of 1000-5000 Da, an operating pressure of 0.1-0.3 MPa, and an operating temperature of 25-30 °C, and collecting the ultrafiltration permeate; The microfiltration step of the crude lecithin extract includes: passing the crude lecithin extract separately through a microfiltration membrane with a pore size of 0.1 to 0.45 μm.

4. The method for extracting crocodile egg yolk lecithin polypeptides according to claim 1, characterized in that, In step S4: The step of adjusting the pH value of the lecithin polypeptide mixture includes: controlling the temperature of the lecithin polypeptide mixture to 30-35°C and adjusting the pH value of the lecithin polypeptide mixture to 7.8-8.2; The step of adding sodium citrate and sucrose fatty acid ester and stirring includes: adding sodium citrate and sucrose fatty acid ester to the lecithin polypeptide mixture after adjusting the pH value, and stirring continuously at a speed of 100-200 r / min; The step of adding polyethylene glycol 400 under ultrasound includes: controlling the ultrasound frequency to 20kHz and the ultrasound power to 150W, adding polyethylene glycol 400 at a uniform rate over 10 minutes under the action of the ultrasound field, and continuing the ultrasound treatment for 15 to 25 minutes. The step of adding sodium alginate and stirring to obtain the modified mixture includes: maintaining the temperature of the lecithin polypeptide mixture at 30-35°C and the pH value at 7.8-8.2, adding sodium alginate at a uniform rate over 5 minutes, and stirring continuously at a speed of 100-200 r / min for 20-30 minutes to obtain the modified mixture.

5. The method for extracting crocodile egg yolk lecithin polypeptides according to claim 1, characterized in that, In step S4: The vacuum concentration is carried out at 40–50°C and a pressure of -0.09–-0.06 MPa. The freeze-drying is carried out at a freezing temperature of -55 to -45°C and a vacuum degree of 10 to 20 Pa for 24 to 36 hours.

6. The method for extracting crocodile egg yolk lecithin polypeptides according to claim 1, characterized in that, In step S4: The average molecular weight of the polyethylene glycol 400 is 380-420; The monoester content of the sucrose fatty acid ester is greater than or equal to 70% by mass. The sodium alginate is a natural polysaccharide with a weight-average molecular weight of 100,000 to 300,000 Da.

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