A method for the combined extraction of multiple components from egg yolk and its application.

Abstract

This invention discloses a method for the combined extraction of multiple components from egg yolk and its application, belonging to the field of food deep processing technology. The method involves centrifuging egg yolk liquid to obtain a slurry and egg yolk particles; the slurry undergoes secondary salting-out to obtain egg yolk immunoglobulin (IgY); the egg yolk particles are pretreated, mixed evenly with an organic solvent, centrifuged, and the precipitate is subjected to a secondary extraction to obtain egg yolk high-phosphorus protein (PV); the supernatant is subjected to low-temperature cold crystallization, centrifugation, and rotary evaporation; the evaporated product is mixed evenly with acetone and centrifuged to obtain precipitated lecithin (PL); the lipid supernatant generated during the extraction process is mixed and rotary evaporated to obtain egg yolk oil. This method achieves the combined extraction of multiple components such as IgY, PV, PL, and egg yolk oil. The process of this invention is simple to operate, low in cost, and has a high raw material utilization rate, making it suitable for large-scale industrial production. The extracted products can be widely used in the food, feed, and biopharmaceutical fields.

Description

Technical Field

[0001] This invention relates to the field of food deep processing technology, and in particular to a method for the combined extraction of multiple components from egg yolk and its application. Background Technology

[0002] Eggs are one of the few globally shared foods, rich in nutrients. Not only is their protein content high, but its amino acid composition is also similar to human protein, making it a high-quality, complete protein, second only to cow's milk, with a bioavailability of up to 95%. The yolk is the most nutritious part of an egg; lipids, fat-soluble vitamins, essential fatty acids, and minerals are found in very small amounts in the egg white, almost entirely in the yolk. The egg yolk contains various components such as egg yolk oil, lecithin (PL), phosphoprotein (PV), and immunoglobulin (IgY), which possess physiological functions including antibacterial, antioxidant, immune-boosting, anti-inflammatory, and anti-cancer properties.

[0003] Currently, most egg yolk extraction processes involve single-component extraction, leading to waste of byproducts and hindering the upgrading of the egg processing industry and the maximization of egg yolk value. For example, if only lecithin or IgY antibodies are extracted, the remaining byproducts containing other active components (such as protein residue and residual lipids) are often discarded or treated as low-cost feed ingredients, resulting in the waste of multiple high-value functional components in the egg yolk. The comprehensive utilization rate of raw materials is typically less than 60%, significantly reducing the overall economic value of the egg yolk. While some multi-component combined extraction technologies for egg yolk exist, they generally suffer from numerous drawbacks. For instance, the purification methods are crude, involving only a single separation, resulting in low product recovery rates. Adding compounds during separation, while improving product purity, often leaves residues that are difficult to remove completely, affecting the compliant application of the product in the food and pharmaceutical fields. Furthermore, solvent recovery and wastewater treatment are challenging; solvents cannot be efficiently recycled and reused, and the discharge of large amounts of waste solvents causes serious environmental pollution. Therefore, to promote the further development of egg processing technology and enhance the overall value of eggs, research on the comprehensive utilization of egg yolk components has become an urgent issue to be addressed. This invention develops a method for the combined extraction of multiple components from egg yolks, providing a production base for the research and development of related functional foods, thereby realizing the comprehensive utilization of egg yolks in the deep processing system of egg products. Summary of the Invention

[0004] The purpose of this invention is to address the current situation where the industrialization of single or multi-component combined extraction of various active ingredients in egg yolk is low, and to provide a method for the combined extraction of multiple components from egg yolk and its application. This study uses poultry eggs, especially chicken egg yolks, as raw material. Based on a comprehensive review of various raw egg yolk component extraction methods, it investigates a method for the combined extraction of multiple components from egg yolk. The aim is to separate the components without damaging the subsequent product components, separating egg yolk immunoglobulins, egg yolk phosphatidylcholine, lecithin, and egg yolk oil. This provides some reference for the industrialization of combined extraction of egg yolk components, maximizes resource utilization, and has certain significance for the comprehensive utilization research of egg yolk.

[0005] To achieve the above objectives, the present invention provides a method for the combined extraction of multiple components from egg yolk, comprising the following steps: S1. Centrifuge the egg yolk liquid to obtain a paste and egg yolk particles; S2. After the slurry described in step S1 is subjected to a first salting-out process, it is centrifuged to obtain precipitate I and supernatant A; the precipitate I is subjected to a second salting-out process and freeze-dried to obtain 1g of Y product; S3. After pre-treating the egg yolk particles described in step S1, mix them evenly with 2 to 10 times the volume of organic solvent and centrifuge to obtain crude PV precipitate and crude PL supernatant. S4. Mix the crude PV precipitate obtained in step S3 with 2-8 times the volume of PV extraction solution, let stand, add 0.5-2 times the volume of PV dissolving solution, adjust the pH to 3-5, and centrifuge to obtain crude PV precipitate and supernatant B1; mix the crude PV precipitate with 2-10 times the volume of deionized water, add 0.5-2 times the volume of PV dissolving solution, adjust the pH to 3-5, and centrifuge to obtain precipitate II and supernatant B2; mix supernatants B1 and B2, heat in a water bath, dialyze, and freeze-dry to obtain the PV product; wherein, the PV extraction solution contains 5wt%-25wt% sodium chloride and 0.01-0.1mol / L sodium hydroxide; S5. The supernatant of the crude PL obtained in step S3 is subjected to low-temperature cold crystallization, and centrifuged to obtain precipitate III and supernatant C; the supernatant C is subjected to rotary evaporation, and the evaporated product is mixed with 2 to 8 times the volume of acetone, allowed to stand, and centrifuged to obtain supernatant D and precipitate IV; the precipitate IV is mixed with 2 to 8 times the volume of acetone, and centrifuged to obtain precipitate PL and supernatant E.

[0006] Further, the process includes step S6: The supernatant A from step S2 and the precipitate II from step S4 are mixed evenly with 2-5 times their volume of organic solvent, and the resulting supernatant is mixed with the precipitate III, supernatant D, and supernatant E from step S5 to obtain a lipid mixture; the lipid mixture is then subjected to rotary evaporation until significant adhesion to the walls is observed, yielding egg yolk oil. Further, the organic solvent is anhydrous ethanol; further, the centrifugation conditions include: centrifugation at 3000-5000 rpm for 5-10 minutes; further, the rotary evaporation conditions include: a temperature of 35-45°C and a rotation speed of 300-500 rpm.

[0007] Further, in step S1: the egg yolk obtained after centrifuging the poultry egg is mixed with deionized water at a mass ratio of 1:2 to 1:10 to obtain an egg yolk liquid; preferably, the egg yolk is pretreated before being mixed with deionized water.

[0008] Furthermore, in step S1: the pretreatment includes: placing the egg yolk on filter paper and rolling it to remove excess egg white adhering to its surface, and then puncturing the yolk membrane.

[0009] Furthermore, in step S1: the preparation conditions of the egg yolk liquid include: stirring at a constant temperature of 20~15℃ and a magnetic stirrer at a speed of 300~500rpm for 1~3 hours to mix evenly, and then refrigerating at 4℃ overnight.

[0010] Furthermore, in step S1, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 10~30 min.

[0011] Further, in step S2: the conditions for the first salting out include: mixing the slurry with ammonium sulfate and sodium chloride solution so that the mixed solution contains 10wt%~30wt% ammonium sulfate and 5wt%~25wt% sodium chloride, and letting it stand overnight at 4°C.

[0012] Furthermore, in step S2, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 10~30 min.

[0013] Further, in step S2: the conditions for the secondary salting out include: mixing precipitate A with sodium chloride and ammonium sulfate solutions so that the mixed solution contains 5wt%~15wt% sodium chloride and 5wt%~20wt% ammonium sulfate, placing it in a 10kDa dialysis bag, and dialyzing it with deionized water at 4℃ for 16~24h, changing the water 4-7 times during the desalination period, and maintaining a vacuum degree of 0.2~0.7Pa.

[0014] Furthermore, in step S2: the freeze-drying conditions include: vacuum drying at a temperature of -60 ~ -40℃ for 24 ~ 48 hours.

[0015] Further, in step S3: the pretreatment includes: mixing egg yolk particles with deionized water at a mass ratio of 1:2 to 1:10, centrifuging at 3000 to 5000 rpm for 10 to 30 minutes, and then collecting the precipitate.

[0016] Furthermore, in step S3: the organic solvent is anhydrous ethanol.

[0017] Furthermore, in step S3, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 5~10 min.

[0018] Further, in step S4: the PV dissolving solution is selected from biological buffer solution or deionized water.

[0019] Furthermore, in step S4, the conditions for settling include 0.5 to 2 hours.

[0020] Furthermore, in step S4, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 10~30 min.

[0021] Furthermore, in step S4, the conditions for water bath heating include: 70~90℃ water bath for 10~30 minutes.

[0022] Further, in step S4: the dialysis conditions include: placing the sample in a 10kDa dialysis bag, dialyzing with deionized water at 4°C for 16-24 hours, changing the water 4-7 times during the desalination period, and maintaining a vacuum of 0.2-0.7 Pa.

[0023] Furthermore, in step S4, the freeze-drying conditions include: vacuum drying at a temperature of -60 ~ -40℃ for 24 ~ 48 hours.

[0024] Furthermore, in step S5, the conditions for low-temperature cold crystallization include placing the crude PL supernatant from step S3 in an environment of -20°C for 10~16 hours.

[0025] Furthermore, in step S5, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 5~10 min.

[0026] Furthermore, in step S5: the conditions for rotary evaporation include: a temperature of 35~45℃, a rotation speed of 300~500rpm, and stopping when the product shows obvious adhesion to the wall.

[0027] A second aspect of the present invention provides the application of the method described herein in food processing, including food ingredients and fortification, food additives or flavorings and oil processing.

[0028] Furthermore, in the application, the purity of egg yolk immunoglobulin (IgY) is ≥90%, the purity of egg yolk high phosphoprotein (PV) is ≥85%, and the purity of lecithin (PL) is ≥80%.

[0029] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides a method for the combined extraction of multiple components from egg yolk, achieving the combined extraction of IgY, PV, PL, and egg yolk oil. The use of water dilution and low-salt stepwise precipitation avoids the damage to the IgY and PV antibody structures caused by high salt concentrations, ensuring the bioactivity of the product. Simultaneously, the low-salt environment reduces the operational costs of subsequent desalting steps. The use of acetone for PL extraction significantly improves PL purity. This invention fully utilizes the active ingredients in the egg yolk raw material, improving raw material utilization and reducing the production cost of single-component extraction. The operation steps are simple and the conditions are mild, requiring no complex equipment, making it suitable for large-scale industrial production. The extracted product has high purity and yield, meeting the application needs of the food, feed, and biopharmaceutical fields. Attached Figure Description

[0030] Figure 1 This is a process flow diagram of an embodiment of the present invention. Detailed Implementation

[0031] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0032] According to one aspect of the present invention, a method for the combined extraction of multiple components from egg yolk is provided, comprising the following steps: S1. Centrifuge the egg yolk liquid to obtain a paste and egg yolk particles; S2. After the slurry described in step S1 is subjected to a first salting-out process, it is centrifuged to obtain precipitate I and supernatant A; the precipitate I is subjected to a second salting-out process and freeze-dried to obtain 1g of Y product; S3. After pre-treating the egg yolk particles described in step S1, mix them evenly with 2 to 10 times the volume of organic solvent and centrifuge to obtain crude PV precipitate and crude PL supernatant. S4. Mix the crude PV precipitate obtained in step S3 with 2-8 times the volume of PV extraction solution, let stand, add 0.5-2 times the volume of PV dissolving solution, adjust the pH to 3-5, and centrifuge to obtain crude PV precipitate and supernatant B1; mix the crude PV precipitate with 2-10 times the volume of deionized water, add 0.5-2 times the volume of PV dissolving solution, adjust the pH to 3-5, and centrifuge to obtain precipitate II and supernatant B2; mix supernatants B1 and B2, heat in a water bath, dialyze, and freeze-dry to obtain the PV product; wherein, the PV extraction solution contains 5wt%-25wt% sodium chloride and 0.01-0.1mol / L sodium hydroxide; S5. The supernatant of the crude PL obtained in step S3 is subjected to low-temperature cold crystallization, and centrifuged to obtain precipitate III and supernatant C; the supernatant C is subjected to rotary evaporation, and the evaporated product is mixed with 2 to 8 times the volume of acetone, allowed to stand, and centrifuged to obtain supernatant D and precipitate IV; the precipitate IV is mixed with 2 to 8 times the volume of acetone, and centrifuged to obtain precipitate PL and supernatant E.

[0033] Further, the process includes step S6: The supernatant A from step S2 and the precipitate II from step S4 are mixed evenly with 2-5 times their volume of organic solvent, and the resulting supernatant is mixed with the precipitate III, supernatant D, and supernatant E from step S5 to obtain a lipid mixture; the lipid mixture is then subjected to rotary evaporation until significant adhesion to the walls is observed, yielding egg yolk oil. Further, the organic solvent is anhydrous ethanol; further, the centrifugation conditions include: centrifugation at 3000-5000 rpm for 5-10 minutes; further, the rotary evaporation conditions include: a temperature of 35-45°C and a rotation speed of 300-500 rpm.

[0034] In this invention, after the egg yolk liquid is centrifuged in step S1, filtration is performed when separating the slurry and particles to prevent the free-flowing particles from flowing out when the slurry is poured out.

[0035] In this invention, during the salting out of the slurry in step S2, the saturated ammonium sulfate solution should be poured in small amounts and slowly multiple times to avoid excessively high salt concentration in any area.

[0036] In this invention, the homogenization in step S3 is carried out at 10,000 rpm for 2 to 4 minutes. Unless otherwise specified, this condition is used for subsequent homogenization. This is only an example and does not limit the scope of the invention.

[0037] In this invention, adding the PV extraction solution in step S4 breaks the calcium phosphate bridge between PV and HDL, allowing PV to be released. The subsequent addition of the PV dissolving solution aims to extract as much PV as possible from the precipitate, thereby increasing the PV extraction efficiency.

[0038] In this invention, precipitate III in step S5 is rich in triglycerides.

[0039] In some embodiments, in step S1: the egg yolk obtained after centrifuging the poultry egg is mixed with deionized water at a mass ratio of 1:2 to 1:10 to obtain an egg yolk liquid; preferably, the egg yolk is pretreated before being mixed with deionized water.

[0040] In some preferred embodiments, in step S1: the pretreatment includes: placing the egg yolk on filter paper and rolling it to remove excess egg white adhering to its surface, and then puncturing the yolk membrane.

[0041] In some preferred embodiments, in step S1, the preparation conditions for the egg yolk liquid include: stirring at a constant temperature of 20-15°C with a magnetic stirrer at a speed of 300-500 rpm for 1-3 hours until homogeneous, and then refrigerating overnight at 4°C. The purpose is to ensure that the egg yolk immunoglobulins are fully dissolved in the water.

[0042] In some preferred embodiments, in step S1, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 10~30 min.

[0043] In some embodiments, in step S2, the conditions for the first salting out include: mixing the slurry with an ammonium sulfate and sodium chloride solution such that the mixed solution contains 10wt%~30wt% ammonium sulfate and 5wt%~25wt% sodium chloride, and letting it stand overnight at 4°C.

[0044] In some embodiments, in step S2, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 10~30 min.

[0045] In some embodiments, in step S2, the conditions for the secondary salting-out include: mixing precipitate A with sodium chloride and ammonium sulfate solutions such that the mixed solution contains 5wt%~15wt% sodium chloride and 5wt%~20wt% ammonium sulfate, placing it in a 10kDa dialysis bag, and dialyzing it with deionized water at 4°C for 16~24h, changing the water 4-7 times during the desalination period, and maintaining a vacuum of 0.2~0.7Pa.

[0046] In some embodiments, in step S2, the freeze-drying conditions include: vacuum drying at a temperature of -60 ~ -40°C for 24 ~ 48 hours.

[0047] In some embodiments, in step S3: the pretreatment includes: mixing egg yolk particles with deionized water at a mass ratio of 1:2 to 1:10, centrifuging at 3000 to 5000 rpm for 10 to 30 minutes, and then collecting the precipitate.

[0048] In some embodiments, in step S3, the organic solvent is anhydrous ethanol.

[0049] In some embodiments, in step S3, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 5~10 min.

[0050] In some embodiments, in step S4: the PV dissolving solution is selected from biological buffer solution or deionized water.

[0051] In some embodiments, in step S4, the conditions for settling include 0.5 to 2 hours.

[0052] In some embodiments, in step S4, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 10~30 min.

[0053] In some embodiments, in step S4, the conditions for water bath heating include: 70~90℃ water bath for 10~30 minutes.

[0054] In some embodiments, in step S4, the dialysis conditions include: placing the sample in a 10kDa dialysis bag and dialyzing with deionized water at 4°C for 16-24 hours, changing the water 4-7 times during the desalination period, and maintaining a vacuum of 0.2-0.7 Pa.

[0055] In some embodiments, in step S4, the freeze-drying conditions include: vacuum drying at a temperature of -60 ~ -40°C for 24 ~ 48 hours.

[0056] In some embodiments, the conditions for low-temperature cold crystallization in step S5 include placing the crude PL supernatant from step S3 in an environment of -20°C for 10 to 16 hours.

[0057] In some embodiments, in step S5, the centrifugation conditions include: centrifugation at 3000~5000 rpm for 5~10 min.

[0058] In some embodiments, in step S5, the conditions for rotary evaporation include: a temperature of 35~45℃, a rotation speed of 300~500rpm, and stopping when the product shows obvious adhesion to the wall.

[0059] A second aspect of the present invention provides the application of the method described herein in food processing, including food ingredients and fortification, food additives or flavorings and oil processing.

[0060] In some embodiments, the purity of egg yolk immunoglobulin (IgY) is ≥90%, the purity of egg yolk phosphatidylcholine (PV) is ≥85%, and the purity of lecithin (PL) is ≥80%. Compared with existing extraction technologies, the products obtained using the above-described methods are free of chemical reagent residues and impurities such as extraneous proteins and triglycerides, fully complying with food-grade safety standards and providing a quality foundation for subsequent food applications.

[0061] The technical solutions of 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, not all, of the embodiments of the present invention. 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.

[0062] Unless otherwise specified, the raw materials used in the following embodiments are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0063] Example 1 This embodiment provides a method for the combined extraction of multiple components from egg yolk, and its process flow diagram is shown below. Figure 1 As shown, the specific steps include: S1: After washing the surface of fresh eggs, crack the shells and separate the egg whites from the yolks. Remove the yolk membrane from the yolks. Mix 600g of egg yolks and ultrapure water at a mass ratio of 1:6 until homogeneous. Stir at 300rpm for 1 hour using a temperature-controlled magnetic stirrer, and let stand overnight at 4℃. Centrifuge at 4000rpm for 15 minutes to obtain a slurry and particles.

[0064] S2: Add 20% ammonium sulfate and 15% sodium chloride to the slurry and let it stand overnight at 4°C. Centrifuge at 4000 rpm for 15 min to collect precipitate I (containing IgY) and supernatant A (containing LDL). Precipitate I undergoes a second salting-out process. Dissolve the precipitate in a fixed concentration of 15% NaCl and a final concentration of 10% saturated ammonium sulfate, and place it in a 10 kDa dialysis bag. Dialyze with ultrapure water at 4°C for 20 h, changing the water 6 times during this period. Freeze-dry to obtain the final IgY product.

[0065] S3: Add egg yolk particles to distilled water at a ratio of 1:2, centrifuge at 4000 rpm for 15 min, and collect the precipitate. Add 4 volumes of anhydrous ethanol, homogenize at 10000 rpm for 4 min, centrifuge at 4000 rpm for 10 min, and collect the coarse PV precipitate and coarse PL supernatant.

[0066] S4: The crude PV precipitate was purified by adding 4 volumes of 0.05 mol / L sodium hydroxide solution containing 20% ​​sodium chloride to dissolve the precipitate, homogenizing at 10,000 rpm for 4 min, and allowing it to stand for 1 h. Then, the same volume of distilled water was added to adjust the pH to 4.0. After centrifugation at 4,000 rpm for 15 min, the precipitate and supernatant B1 were collected. The precipitate was extracted again by adding 5 volumes of distilled water and homogenizing at 10,000 rpm for 4 min. Then, 1 volume of distilled water was added to adjust the pH to 4.0. After centrifugation, the supernatant B2 and precipitate II were collected. B1 and B2 were mixed and placed in an 80℃ water bath for 20 min. The mixture was then dialyzed with ultrapure water at 4℃ for 20 h to desalinate, with the water changed 6 times during the process. Finally, the mixture was freeze-dried to obtain the PV product.

[0067] S5: The supernatant of crude PL was cryogenically crystallized and placed at -20℃ overnight. It was then centrifuged at 4000 rpm for 10 min. Precipitate III was triglyceride, and supernatant C was collected. Supernatant C was rotary evaporated at 40℃ and 300 rpm until the product showed obvious adhesion to the wall. 4 volumes of acetone were added to the evaporated product and mixed well. The mixture was centrifuged at 4000 rpm for 15 min to obtain supernatant D and precipitate IV. Acetone was added to precipitate IV again and mixed well. The mixture was centrifuged at 4000 rpm for 15 min, and precipitate (PL) and supernatant E were collected.

[0068] S6: Add 2g of anhydrous ethanol to supernatant A and precipitate II, mix well, centrifuge at 4000rpm for 10min, take the supernatant of each, mix each supernatant with precipitate III, supernatant D and supernatant E to obtain lipid mixture, and then perform rotary evaporation at 40℃ and 300rpm. Stop when there is obvious adhesion to the wall to obtain egg yolk oil.

[0069] The yield and purity of the obtained components are shown below: Example 2 This embodiment provides a method for the combined extraction of multiple components from egg yolk, and its process flow diagram is shown below. Figure 1 As shown, the specific steps include: S1: After washing the surface of fresh eggs, crack the shells and separate the egg whites from the yolks. Remove the yolk membrane from the yolks. Mix 600g of egg yolks and ultrapure water at a mass ratio of 1:4 until homogeneous. Stir at 400rpm for 1.5 hours using a temperature-controlled magnetic stirrer and let stand overnight at 4℃. Centrifuge at 4500rpm for 10 minutes to obtain a slurry and particles.

[0070] S2: Add 15% ammonium sulfate and 10% sodium chloride to the slurry and let it stand overnight at 4°C. Centrifuge at 4500 rpm for 10 min to collect precipitate I (containing IgY) and supernatant A (containing LDL). Precipitate I undergoes secondary salting out. Dissolve the precipitate in a fixed concentration of 5% NaCl and a final concentration of 15% saturated ammonium sulfate, and place it in a 10 kDa dialysis bag. Dialyze with ultrapure water at 4°C for 22 h, changing the water 5 times during the process. Freeze-dry to obtain the final IgY product.

[0071] S3: Add egg yolk particles to distilled water at a ratio of 1:6, centrifuge at 4500 rpm for 10 min, and collect the precipitate. Add 2 volumes of anhydrous ethanol, homogenize at 10000 rpm for 2 min, centrifuge at 4500 rpm for 5 min, and collect the coarse PV precipitate and coarse PL supernatant.

[0072] S4: The crude PV precipitate was purified by adding 6 volumes of 0.03 mol / L sodium hydroxide solution containing 10% sodium chloride to dissolve the precipitate, homogenizing at 10,000 rpm for 2 min, and allowing it to stand for 0.5 h. Then, the same volume of distilled water was added to adjust the pH to 4.5. After centrifugation at 4500 rpm for 10 min, the precipitate and supernatant B1 were collected. The precipitate was extracted again by adding 5 volumes of distilled water and homogenizing at 10,000 rpm for 2 min. Then, 1 volume of distilled water was added to adjust the pH to 4.5. After centrifugation, the supernatant B2 and precipitate II were collected. B1 and B2 were mixed and placed in a water bath at 75°C for 25 min. The mixture was then dialyzed with ultrapure water at 4°C for 22 h to desalinate, with the water changed 5 times during the process. Finally, the mixture was freeze-dried to obtain the PV product.

[0073] S5: The supernatant of crude PL was cryogenically crystallized at low temperature and placed at -20℃ overnight. It was centrifuged at 4500rpm for 5min. Precipitate III was triglyceride. The supernatant C was collected. The supernatant C was rotary evaporated at 35℃ and 400rpm. The process was stopped when the product showed obvious adhesion to the wall. Two volumes of acetone were added to the evaporated product and mixed evenly. The mixture was centrifuged at 4500rpm for 10min to obtain the supernatant and precipitate IV. Acetone was added to precipitate IV again and mixed evenly. The mixture was centrifuged at 4500rpm for 10min. The precipitate (PL) and supernatant E were collected. The precipitate was freeze-dried to obtain the PL product.

[0074] S6: Add 3g of anhydrous ethanol to supernatant A and precipitate II, mix well, centrifuge at 4500rpm for 5min, take the supernatant of each, mix each supernatant with precipitate III, supernatant D and supernatant E to obtain lipid mixture, and then perform rotary evaporation at 35℃ and 400rpm. Stop when there is obvious adhesion to the wall to obtain egg yolk oil.

[0075] The yield and purity of the obtained components are shown below: Example 3 This embodiment provides a method for the combined extraction of multiple components from egg yolk, and its process flow diagram is shown below. Figure 1 As shown, the specific steps include: S1: After washing the surface of fresh eggs, crack the shells and separate the egg whites from the yolks. Remove the yolk membrane from the yolks. Mix 600g of egg yolks and ultrapure water at a mass ratio of 1:10 until homogeneous. Stir at 500rpm for 2 hours using a constant temperature magnetic stirrer, and let stand overnight at 4℃. Centrifuge at 3800rpm for 20 minutes to obtain a slurry and particles.

[0076] S2: Add 25% ammonium sulfate and 20% sodium chloride to the slurry and let it stand overnight at 4°C. Centrifuge at 3800 rpm for 20 min to collect precipitate I (containing IgY) and supernatant A (containing LDL). Precipitate I undergoes secondary salting out. Dissolve the precipitate in a fixed concentration of 15% NaCl and a final concentration of 20% saturated ammonium sulfate, and place it in a 10 kDa dialysis bag. Dialyze with ultrapure water at 4°C for 24 h, changing the water 7 times during this period. Freeze-dry to obtain the final IgY product.

[0077] S3: Add egg yolk particles to distilled water at a ratio of 1:4, centrifuge at 3800 rpm for 20 min, and collect the precipitate. Add 8 volumes of anhydrous ethanol, homogenize at 10000 rpm for 3 min, centrifuge at 3800 rpm for 6 min, and collect the coarse PV precipitate and coarse PL supernatant.

[0078] S4: The crude PV precipitate was purified by adding 8 volumes of 0.01 mol / L sodium hydroxide solution containing 15% sodium chloride to dissolve the precipitate, homogenizing at 10,000 rpm for 3 min, and allowing it to stand for 1 h. Then, the same volume of distilled water was added to adjust the pH to 4.3. After centrifugation at 3800 rpm for 20 min, the precipitate and supernatant B1 were collected. The precipitate was extracted again by adding 5 volumes of distilled water and homogenizing at 10,000 rpm for 3 min. Then, 1 volume of distilled water was added to adjust the pH to 4.3. After centrifugation, the supernatant B2 and precipitate II were collected. B1 and B2 were mixed and placed in a water bath at 70℃ for 30 min. The mixture was then dialyzed with ultrapure water at 4℃ for 24 h to remove salt, with the water changed 7 times during the process. Finally, the mixture was freeze-dried to obtain the PV product.

[0079] S5: The crude PL supernatant was used to cold crystallize the egg yolk ethanol extract (containing PL) at low temperature and placed at -20℃ overnight. It was then centrifuged at 3800 rpm for 10 min. Precipitate III was triglycerides, and the supernatant C was collected. Supernatant C was rotary evaporated at 45℃ and 500 rpm until the product showed obvious adhesion to the wall. 6 volumes of acetone were added to the evaporated product and mixed well. The mixture was then centrifuged at 3800 rpm for 20 min to obtain the supernatant and precipitate IV. Acetone was added to precipitate IV again and mixed well. The mixture was then centrifuged at 3800 rpm for 20 min, and the precipitate (PL) and supernatant E were collected.

[0080] S6: Add 4g of anhydrous ethanol to supernatant A and precipitate II, mix well, centrifuge at 3800rpm for 6min, take the supernatant of each, mix each supernatant with precipitate III, supernatant D and supernatant E to obtain lipid mixture, and then perform rotary evaporation at 45℃ and 500rpm. Stop when there is obvious adhesion to the wall to obtain egg yolk oil.

[0081] The yield and purity of the obtained components are shown below: Comparative Example 1 The difference from Example 1 is that a single salting-out method was used to extract IgY. Only 15% ammonium sulfate was added to the egg yolk slurry, and after standing overnight at 4°C, the precipitate was collected by centrifugation at 3800 rpm. There was no 10% sodium chloride co-salting-out step or secondary salting-out step. The precipitate was directly dialyzed for 12 hours (with 3 water changes) using a 5kDa dialysis bag and then freeze-dried. This process resulted in severe contamination of other proteins, and the IgY purity was only 72%~76%, far lower than the ≥90% purity obtained by this method after secondary salting-out and 20 hours of dialyzed with a 10kDa dialysis bag (with 6-7 water changes).

[0082] Comparative Example 2 The difference from Example 1 is that IgY was extracted using the PEG6000 precipitation method. A final concentration of 8% PEG6000 was added to the slurry, the precipitate was collected by centrifugation, and then reconstituted with physiological saline. Only one dialysis desalination was performed. This process resulted in difficult removal of PEG residues and no secondary purification step, achieving an IgY purity of 70%–75%.

[0083] Comparative Example 3 The difference from Example 1 is that a single-stage alkaline precipitation method was used to extract PV. 0.05 mol / L sodium hydroxide was added to the yolk granule precipitate for dissolution, the pH was directly adjusted to 4.0, and the supernatant was collected by centrifugation. There was no 15% sodium chloride to aid dissolution or a secondary extraction step, and the supernatant was not subjected to 80°C water bath heat purification. This process resulted in insufficient PV dissolution, ineffective removal of impurities, and a purity of only 69%–74%, lower than the ≥85% purity achieved by this method.

[0084] Comparative Example 4 The difference from Example 1 is that a high-salt extraction method was used to extract PV. A 40% sodium chloride solution was added to the egg yolk particles for homogenization and extraction. After centrifugation, the supernatant was adjusted to pH 4.0 to precipitate PV. The precipitate was collected after only one centrifugation and then freeze-dried. This process eliminates the need for alkali solution optimization and stepwise dilution to adjust pH, resulting in a PV purity of 71%–76%.

[0085] Comparative Example 5 The difference from Example 1 is that PL is extracted using a single ethanol extraction method. Two volumes of anhydrous ethanol are added to the egg yolk particles, homogenized, centrifuged, and the supernatant is directly evaporated by rotary evaporation and then freeze-dried, without the low-temperature cold crystallization to remove triglycerides and the acetone washing step. This process results in high levels of residual triglycerides and cholesterol, with PL purity only 62%~67%, far lower than the ≥80% purity achieved by this method after two acetone washing steps.

[0086] Comparative Example 6 The difference from Example 1 is that PL is extracted using a hexane-ethanol mixed solvent, and the supernatant is concentrated by rotary evaporation after extraction, followed by only one acetone precipitation purification. This process uses a mixed solvent that easily carries impurities and lacks a secondary washing step, resulting in a PL purity of 65%–70%, but with a higher solvent residue than the method described above.

[0087] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A method for the combined extraction of multiple components from egg yolk, characterized in that, Includes the following steps: S1. Centrifuge the egg yolk liquid to obtain a paste and egg yolk particles; S2. After the slurry described in step S1 is subjected to a first salting-out process, it is centrifuged to obtain precipitate I and supernatant A; the precipitate I is subjected to a second salting-out process and freeze-dried to obtain 1g of Y product; S3. After pre-treating the egg yolk particles described in step S1, mix them evenly with 2 to 10 times the volume of organic solvent and centrifuge to obtain crude PV precipitate and crude PL supernatant. S4. Mix the crude PV precipitate obtained in step S3 with 2-8 times the volume of PV extraction solution, let stand, add 0.5-2 times the volume of PV dissolving solution, adjust the pH to 3-5, and centrifuge to obtain crude PV precipitate and supernatant B1; mix the crude PV precipitate with 2-10 times the volume of deionized water, add 0.5-2 times the volume of PV dissolving solution, adjust the pH to 3-5, and centrifuge to obtain precipitate II and supernatant B2; mix supernatants B1 and B2, heat in a water bath, dialyze, and freeze-dry to obtain the PV product; wherein, the PV extraction solution contains 5wt%-25wt% sodium chloride and 0.01-0.1mol / L sodium hydroxide; S5. The supernatant of the crude PL obtained in step S3 is subjected to low-temperature cold crystallization, and centrifuged to obtain precipitate III and supernatant C; the supernatant C is subjected to rotary evaporation, and the evaporated product is mixed with 2 to 8 times the volume of acetone, allowed to stand, and centrifuged to obtain supernatant D and precipitate IV; the precipitate IV is mixed with 2 to 8 times the volume of acetone, and centrifuged to obtain precipitate PL and supernatant E.

2. The method according to claim 1, characterized in that, The process also includes step S6: mixing the supernatant A from step S2 and the precipitate II from step S4 with 2 to 5 times the volume of organic solvent, centrifuging the resulting supernatant, and mixing it with the precipitate III, supernatant D, and supernatant E from step S5 to obtain a lipid mixture; and then rotary evaporating the lipid mixture until there is obvious adhesion to the wall to obtain egg yolk oil.

3. The method according to claim 2, characterized in that, The organic solvent is anhydrous ethanol; and / or The centrifugation conditions include: centrifugation at 3000~5000 rpm for 5~10 min; and / or The conditions for rotary evaporation include: a temperature of 35~45℃ and a rotation speed of 300~500rpm.

4. The method according to claim 1, characterized in that, In step S1: Egg yolk obtained by centrifuging poultry eggs is mixed with deionized water at a mass ratio of 1:2 to 1:10 to prepare egg yolk liquid. Preferably, the egg yolks are pretreated before being mixed with deionized water; More preferably, The pretreatment includes: rolling the egg yolk on filter paper to remove excess egg white adhering to its surface, and then puncturing the yolk membrane; and / or The preparation conditions for the egg yolk mixture include: stirring at a constant temperature of 20-15℃ and a magnetic stirrer at a speed of 300-500 rpm for 1-3 hours until homogeneous, and then refrigerating overnight at 4℃; and / or The centrifugation conditions include: centrifugation at 3000~5000 rpm for 10~30 min.

5. The method according to claim 1, characterized in that, In step S2: The conditions for the first salting-out include: mixing the slurry with an ammonium sulfate and sodium chloride solution such that the mixed solution contains 10wt%~30wt% ammonium sulfate and 5wt%~25wt% sodium chloride, and letting it stand overnight at 4°C; and / or The centrifugation conditions include: centrifugation at 3000~5000 rpm for 10~30 min; and / or The conditions for the secondary salting-out include: mixing precipitate A with sodium chloride and ammonium sulfate solutions to form a mixed solution containing 5wt%~15wt% sodium chloride and 5wt%~20wt% ammonium sulfate, placing it in a 10kDa dialysis bag, and dialyzing it with deionized water at 4°C for 16~24 hours, changing the water 4~7 times during the desalting period, and maintaining a vacuum of 0.2~0.7 Pa; and / or The freeze-drying conditions include: vacuum drying at a temperature of -60 ~ -40℃ for 24 ~ 48 hours.

6. The method according to claim 1, characterized in that, In step S3: The pretreatment includes: mixing egg yolk particles with deionized water at a mass ratio of 1:2 to 1:10, centrifuging at 3000 to 5000 rpm for 10 to 30 minutes, and collecting the precipitate; and / or The organic solvent is anhydrous ethanol; and / or The centrifugation conditions include: centrifugation at 3000~5000 rpm for 5~10 min.

7. The method according to claim 1, characterized in that, In step S4: The PV dissolving solution is selected from biological buffer solution or deionized water; and / or The conditions for settling include: 0.5~2 hours; and / or The centrifugation conditions include: centrifugation at 3000~5000 rpm for 10~30 min; and / or The conditions for water bath heating include: 70~90℃ water bath for 10~30 min; and / or The dialysis conditions include: placing the sample in a 10 kDa dialysis bag and dialyzing with deionized water at 4°C for 16-24 hours, changing the water 4-7 times during desalination, and maintaining a vacuum of 0.2-0.7 Pa; and / or The freeze-drying conditions include: vacuum drying at a temperature of -60 ~ -40℃ for 24 ~ 48 hours.

8. The method according to claim 1, characterized in that, In step S5: The conditions for low-temperature cold crystallization include: placing the crude PL supernatant from step S3 in an environment of -20°C for 10~16 hours; and / or The centrifugation conditions include: centrifugation at 3000~5000 rpm for 5~10 min; and / or The conditions for rotary evaporation include: a temperature of 35~45℃, a rotation speed of 300~500rpm, and stopping when the product shows obvious adhesion to the wall.

9. The application of the method according to any one of claims 1-8 in food processing, characterized in that, This includes food ingredients and fortification, food additives or flavorings, and oil processing.

10. The application according to claim 9, characterized in that, In the application described, the purity of egg yolk immunoglobulin is ≥90%, the purity of egg yolk high-phosphorus protein is ≥85%, and the purity of lecithin is ≥80%.

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