Eggshell peptide extraction method based on enzyme immobilization technology and application of eggshell peptide

Abstract

The invention discloses an eggshell peptide extraction method based on an enzyme immobilization technology and application of eggshell peptide, and relates to the technical field of biology. S1, egg shells with membranes are smashed; s2, preparing an immobilized enzyme; s3, enzymolysis extraction: suspending the crushed egg shells in a phosphate buffer solution, adding immobilized enzyme according to the mass ratio of the immobilized enzyme to the egg shells being (0.8-1.2): 10, carrying out enzymolysis for 1.8-2.2 hours at 35-38 DEG C, and continuously stirring during enzymolysis; s4, after the enzymolysis reaction is finished, centrifuging to obtain supernate, concentrating and purifying the supernate through an ultrafiltration device to obtain a protein solution, and performing vacuum freeze drying to obtain solid protein powder, namely the eggshell peptide. The method is simple and efficient, the extracted protein has high biological activity, the method is suitable for industrial application, high-value utilization of the egg shells is achieved, and the utilization rate of raw materials is increased.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an eggshell peptide extraction method based on enzyme immobilization technology and application of the eggshell peptide. Background Art

[0002] Eggs are one of the most consumed foods in the world, and a huge amount of eggshells are produced every year. Traditionally, eggshells are often regarded as agricultural waste, and improper disposal will burden the environment. Therefore, turning eggshells into useful resources not only reduces waste but also realizes a circular economy.

[0003] Eggshells are mainly composed of calcium carbonate, accounting for more than 80% of their weight, and contain more than 3% protein, as well as other trace elements. This natural chemical composition provides a material basis for the reuse of eggshells. After the protein in the eggshell is extracted, it can be used as a raw material in the food industry, medicine or cosmetics industry. For example, protein hydrolysate can be used as a nutritional supplement or for the development of new bioactive peptides.

[0004] At present, the large amount of discarded eggshells not only causes a waste of resources, but also may burden the environment. The existing technologies for extracting bioactive peptides from eggshells are mainly physical methods, chemical methods and biological fermentation methods, which have problems such as low extraction efficiency, high cost and environmental pollution. Some innovative extraction technologies, such as sequential extraction using guanidine hydrochloride and EDTA, and methods such as changing the precipitation rate and crystal morphology of calcium carbonate through in vitro experiments, may be difficult to promote on a large scale due to expensive equipment or complex operation and high professional requirements for technicians, affecting their economic efficiency. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an eggshell peptide extraction method based on enzyme immobilization technology and the application of eggshell peptides. Through innovative extraction technology, not only the environmental problems of eggshell treatment are solved, but also a new source of raw materials is opened up, which has significant social, economic and environmental benefits.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for extracting eggshell peptides based on enzyme immobilization technology, comprising:

[0008] S1. Crush the egg shell with membrane;

[0009] S2, preparing immobilized enzyme: dissolving neutral protease in phosphate buffer, mixing with inert carrier solution, stirring to obtain a mixed solution; dropping the mixed solution into CaCl2 solution, stirring to obtain a small spherical product, filtering and washing, adding the small spherical product into glutaraldehyde solution for cross-linking reaction, filtering, washing and drying after the reaction to obtain neutral protease, i.e., immobilized enzyme;

[0010] S3, enzymatic extraction: suspend the crushed eggshell in phosphate buffer, add immobilized enzyme at a mass ratio of 0.8-1.2:10 of enzyme to eggshell, perform enzymatic hydrolysis for 1.8-2.2h at 35-38℃, and stir continuously during the enzymatic hydrolysis;

[0011] S4. After the enzymatic hydrolysis reaction is completed, the supernatant is obtained by centrifugation, and the supernatant is concentrated and purified by an ultrafiltration device to obtain a protein solution, which is then freeze-dried in vacuum to obtain a solid protein powder, i.e., eggshell peptide.

[0012] Furthermore, in step S1, the egg shell with membrane is crushed to 50-100 μm.

[0013] Further, in step S2, 0.4-0.6 g of neutral protease is dissolved in 100 mL of phosphate buffer with a pH of 7.2 to 7.8, and then mixed with an inert carrier solution at a mass ratio of 1:5, and stirred for 0.8-1.2 hours to obtain a mixed solution; the mixed solution is dropped into 50-60 mL of CaCl2 solution from a height of 8-12 cm, stirred at 24-26° C. to obtain a small spherical product, which is filtered and washed with distilled water; the small spherical product is added to 40-60 mL of glutaraldehyde solution, cross-linked at 35-38° C. for 1.5-2.5 hours, and the immobilized enzyme is obtained after filtering, washing and low-temperature drying.

[0014] Furthermore, the inert carrier solution is agarose microsphere or silica microsphere solution with a mass concentration of 4%; the mass concentration of the CaCl2 solution is 2%; and the mass concentration of the glutaraldehyde solution is 0.5%.

[0015] Furthermore, it is characterized in that in step S3, the pH value of the phosphate buffer is 7.2-7.8, and the crushed eggshell is suspended in the phosphate buffer at a concentration of 95-105 g / L.

[0016] Furthermore, in step S3, after each enzymatic hydrolysis reaction is completed, the immobilized enzyme is separated from the solution by filtration or centrifugation, and the immobilized enzyme can be recycled for 5-10 times.

[0017] Furthermore, in step S4, the centrifugation conditions are: 3800-4000 rpm for 12-16 min; and the ultrafiltration membrane in the ultrafiltration device has a molecular weight cutoff of 10 kDa.

[0018] Application of the eggshell peptide extracted by the above extraction method in facial cream.

[0019] Further, the components of the facial cream include water phase ingredients, oil phase ingredients and cooling stage added ingredients;

[0020] Water phase ingredients: 150-170 parts of distilled water, 3.5-4.5 parts of glycerin, 1.5-2.5 parts of colloidal oatmeal;

[0021] Oil phase ingredients: 5-7 parts avocado butter, 16-20 parts grape seed oil, 9-11 parts emulsifying wax;

[0022] Ingredients added during the cooling stage: 4.5-5.5 parts of extracted eggshell peptides, 1.8-2.2 parts of phenoxyethanol, 1.8-2.2 parts of vitamin E, and 0.8-1.2 parts of vanilla essential oil.

[0023] Furthermore, the preparation method of the facial cream is: heating the water phase component and the oil phase component at a temperature of 70-80°C respectively until they are completely melted, mixing the two phases, stirring with a stirrer at 3000-3500rpm for 3-5min to form an emulsion, and after the emulsion is cooled, adding the ingredients added in the cooling stage and mixing well to obtain the facial cream.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention uses immobilized enzyme technology to immobilize the protease on an inert carrier, which can be reused multiple times, thereby improving the extraction efficiency and reducing the amount of enzyme used; in addition, the extracted protein is applied to the preparation of facial cream, showing excellent moisturizing and antioxidant properties, and is particularly suitable for beauty and skin care products. The method of the present invention is simple and efficient, the extracted protein has high biological activity, is suitable for industrial application, realizes the high-value utilization of eggshells, and improves the utilization rate of raw materials.

[0026] (2) The eggshell peptides are extracted by the method provided by the present invention, and the use of harmful chemicals is avoided during the extraction process, thereby reducing the pollution to the environment and meeting the requirements of green chemistry.

[0027] (3) The present invention utilizes widely available egg shells as raw materials, thereby reducing production costs and improving the market competitiveness of products; egg shells that were originally treated as waste are transformed into beneficial resources, thereby increasing the added value of egg shells.

[0028] (4) The calcium carbonate and protein extracted by the present invention have high purity and can meet the needs of different industries for high-quality raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the eggshell peptide extraction process;

[0030] Figure 2 This is a chart showing the changes in moisturizing properties of facial cream;

[0031] Figure 3 DPPH free radical scavenging rate for facial cream. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention include but are not limited to the following embodiments.

[0033] Example 1

[0034] like Figure 1 As shown, this embodiment discloses a method for extracting eggshell peptides based on enzyme immobilization technology, and the specific operations are as follows:

[0035] (1) Pretreatment of egg shell and egg shell membrane: 100 g of egg shell with membrane was mechanically crushed into fine particles of 75 μm at low temperature.

[0036] (2) Preparation of immobilized enzyme: 0.5 g of neutral protease was dissolved in 100 mL of phosphate buffer at pH 7.5, and then mixed with an inert carrier (agarose microsphere) solution with a mass concentration of 4% at a mass ratio of 1:5, and slowly stirred for 1 hour to fix the enzyme molecules on the surface of the carrier through covalent bonds or electrostatic adsorption to obtain a mixed solution. The mixed solution was drawn up with a syringe and slowly dripped into 50 mL of 2% (w%) CaCl2 solution from a height of 10 cm, and slowly stirred at 25°C to solidify to obtain a small spherical product, which was filtered and washed with distilled water; the small spherical product was added to 50 mL of 0.5% (w%) glutaraldehyde solution, cross-linked at 37°C for 2 hours, filtered, washed and low-temperature dried to obtain solid granular immobilized neutral protease, i.e., immobilized enzyme.

[0037] (3) Enzymatic extraction: 10 g of immobilized enzyme was added to 1 liter of pH 7.5 buffer, and 100 g of ground eggshell was added. The enzymatic reaction was accelerated by microwave-assisted heating (2450 MHz, 100 W) at 37 °C. The enzymatic reaction lasted for 2 h, and stirring was maintained during the reaction to promote protein release. The immobilization rate of neutral protease using agarose microspheres as a carrier reached 90%.

[0038] After each enzymatic hydrolysis reaction, the immobilized enzyme is separated from the protein solution by simple filtration or centrifugation. The immobilized enzyme can be reused in the next round of reaction and can be stably used for 5-10 times without a significant decrease in enzyme activity. After testing, the enzyme activity of the immobilized enzyme remained above 80% after being reused 5 times.

[0039] (4) Eggshell peptide purification and freeze-drying: After the enzymatic hydrolysis reaction is completed, centrifuge at 4000rpm for 15min to obtain a supernatant. The supernatant is concentrated and purified by an ultrafiltration device, and an ultrafiltration membrane with a molecular weight cutoff of 10kDa is used to remove small molecule impurities and residual enzymes to obtain a high-purity protein solution. The purified protein solution is vacuum freeze-dried to obtain 9.98g of high-purity solid protein powder, i.e., eggshell peptide, for long-term storage and application. The finished protein extract was identified by LC-MS by a quality inspection company (the test results are shown in Tables 1 and 2), and the protein purity was 73%.

[0040] Table 1

[0041]

[0042] Table 2

[0043]

[0044] The extracted eggshell peptide can be used for the preparation of facial cream, and the components of the facial cream include component 1 (water phase component), component 2 (oil phase component) and component 3 (component added in the cooling stage).

[0045] Component 1 (aqueous phase): 160 g distilled water, 4 g glycerin, 2 g colloidal oatmeal;

[0046] Component 2 (oil phase ingredients): avocado butter 6g, grape seed oil 18g, emulsifying wax 10g;

[0047] Component 3 (ingredients added during the cooling stage): 5 g of extracted eggshell peptide, 2 g of phenoxyethanol, 2 g of vitamin E, and 1 g of vanilla essential oil.

[0048] Preparation of facial cream: Use a double pot to heat the water phase and oil phase ingredients separately at a temperature of 75°C until they are completely melted. Mix the two phases and stir with a stirrer at 3000rpm for 5 minutes to form an emulsion. After the emulsion is cooled, add the ingredients in the cooling stage and mix well. Store the finished facial cream in a dark container to prevent the active ingredients from being damaged by light. In this embodiment, heating the water phase and the oil phase separately can ensure the complete dissolution and uniform distribution of each ingredient. Heating the water phase and the oil phase to similar temperatures and then mixing them can effectively reduce the stratification or unevenness caused by the temperature difference, thereby forming a stable emulsified system.

[0049] The eggshell peptide cream was tested as follows:

[0050] 1. Verification of the moisturizing activity of eggshell peptide cream

[0051] The saturated sodium carbonate solution was placed in a desiccator at room temperature (25±1)°C to control the relative humidity in the desiccator to be 43%. Accurately weigh 1g of each of the creams in experimental group A (commercially available cream), experimental group B (cream without eggshell peptide), and experimental group C (cream with eggshell peptide), and add them to beakers with a diameter of 3cm, respectively, and then placed in the desiccator. The mass M1 before placement was measured, and then the mass M2 of the sample after placement was measured at different times such as 2h, 4h, and 6h, and the moisture absorption rate was calculated, and the moisture absorption rate was plotted into a curve, such as Figure 2 shown.

[0052] from Figure 2 It can be seen that the moisturizing rates of the three experimental groups of creams all showed a downward trend over time. As time changes, the moisturizing rate of the cream without eggshell peptides decreases the fastest, and the moisturizing rate is 66.9% after 6 hours. The moisturizing rate of the cream with eggshell peptides decreases slightly more than the market-sold cream every hour, but the moisturizing rate curves of the two are basically the same, and the moisturizing rate of the developed eggshell peptide cream is still stable at more than 75% after 6 hours, which fully demonstrates that the eggshell peptide cream has good moisturizing ability.

[0053] 2. Verification of the antioxidant activity of eggshell peptide cream

[0054] Use 50% methanol solution to prepare 5g / L, 10g / L, 15g / L, 20g / L, 25g / L eggshell peptide cream solution, add 2mL DPPH solution, place in the dark for 20min, and measure the absorbance at 517nm wavelength. Use 50% methanol solution instead of DPPH solution to perform background test. Use Vc solution as positive control to calculate DPPH clearance rate.

[0055] Clearance = [1-(A1-A2) / A0] × 100%

[0056] Where: A0 is the absorbance of pure water + DPPH solution, A1 is the absorbance of sample + DPPH solution, and A2 is the absorbance of sample + methanol.

[0057] Draw a DPPH clearance curve, such as Figure 3 As shown, it can be seen that with the continuous increase in concentration, the DPPH radical scavenging rate of eggshell peptide cream shows a trend of first rising and then stabilizing. When the sample concentration is about 20%, there is an optimal scavenging concentration. The DPPH radical scavenging rate of Vc is always stable at about 93%. The highest scavenging rate of eggshell peptide cream solution is 55.6%, which is lower than that of Vc solution, but still has strong in vitro antioxidant capacity.

[0058] Example 2

[0059] This example discloses a method for extracting eggshell peptides based on enzyme immobilization technology, and the specific operation is as follows:

[0060] (1) Pretreatment of eggshell and eggshell membrane: The eggshell with membrane is mechanically crushed into fine particles of 80 μm at low temperature.

[0061] (2) Preparation of immobilized enzyme: 0.4 g of neutral protease was dissolved in 100 mL of phosphate buffer at pH 7.2, and then mixed with an inert carrier (agarose microsphere) solution with a mass concentration of 4% at a mass ratio of 1:5, and slowly stirred for 0.8 h to fix the enzyme molecules on the surface of the carrier through covalent bonds or electrostatic adsorption to obtain a mixed solution. The mixed solution was drawn up with a syringe and slowly dripped into 50 mL of 2% (w%) CaCl2 solution from a height of 8 cm, and slowly stirred at 24°C to solidify to obtain a small spherical product, which was filtered and washed with distilled water; the small spherical product was added to 40 mL of 0.5% (w%) glutaraldehyde solution, cross-linked at 35°C for 1.5 h, and solid granular immobilized neutral protease was obtained after filtering, washing and low-temperature drying.

[0062] (3) Enzymatic extraction: The crushed eggshells were suspended in a phosphate buffer solution with a pH of 7.2 at a concentration of 95 g / L. The immobilized neutral protease prepared in advance was added at a weight ratio of 1:10 between the enzyme and the eggshell. The enzymatic reaction was accelerated by microwave-assisted heating (2450 MHz, power of 100 W) at 35 °C. The enzymatic reaction lasted for 2 h, and stirring was maintained during the reaction to promote the release of protein.

[0063] (4) Eggshell peptide purification and freeze-drying: After the enzymatic hydrolysis reaction is completed, the supernatant is obtained by centrifugation at 3800rpm for 14min. The supernatant is concentrated and purified by an ultrafiltration device, and an ultrafiltration membrane with a molecular weight cutoff of 10kDa is used to remove small molecule impurities and residual enzymes to obtain a high-purity protein solution. The purified protein solution is vacuum freeze-dried to obtain a solid protein powder, i.e., eggshell peptide, with a protein purity of 74%, for long-term storage and application.

[0064] The extracted eggshell peptide can be used for the preparation of facial cream, and the components of the facial cream include component 1 (water phase component), component 2 (oil phase component) and component 3 (component added in the cooling stage).

[0065] Component 1 (aqueous phase): 150 g distilled water, 3.5 g glycerol, 1.5 g colloidal oatmeal;

[0066] Component 2 (oil phase ingredients): avocado butter 5g, grape seed oil 16g, emulsifying wax 9g;

[0067] Component 3 (ingredients added during the cooling stage): 4.5 g of extracted eggshell peptide, 1.8 g of phenoxyethanol, 1.8 g of vitamin E, and 0.8 g of vanilla essential oil.

[0068] Preparation of the cream: Heat the water phase and oil phase ingredients separately at 75°C in a double boiler until they are completely melted. Mix the two phases and stir them at 3500rpm for 3 minutes to form an emulsion. After the emulsion cools, add the ingredients from the cooling stage and mix well. Store the finished cream in a dark container to prevent the active ingredients from being destroyed by light.

[0069] Example 3

[0070] This example discloses a method for extracting eggshell peptides based on enzyme immobilization technology, and the specific operation is as follows:

[0071] (1) Pretreatment of eggshell and eggshell membrane: The eggshell with membrane was mechanically crushed into fine particles of 100 μm at low temperature.

[0072] (2) Preparation of immobilized enzyme: 0.6 g of neutral protease was dissolved in 100 mL of phosphate buffer at pH 7.8, and then mixed with an inert carrier (silica gel microsphere) solution with a mass concentration of 4% at a mass ratio of 1:5, and slowly stirred for 1.2 h to fix the enzyme molecules on the carrier surface through covalent bonds or electrostatic adsorption to obtain a mixed solution. The mixed solution was drawn up with a syringe and slowly dripped into 60 mL of 2% (w%) CaCl2 solution from a height of 12 cm, and slowly stirred at 26°C to solidify to obtain a small spherical product, which was filtered and washed with distilled water; the small spherical product was added to 60 mL of 0.5% (w%) glutaraldehyde solution, cross-linked at 38°C for 2.5 h, and solid granular immobilized neutral protease was obtained after filtering, washing and low-temperature drying.

[0073] (3) Enzymatic extraction: The crushed eggshells were suspended in a phosphate buffer solution at pH 7.8 at a concentration of 100 g / L. The immobilized neutral protease prepared in advance was added at a weight ratio of enzyme to eggshell of 1:10. The enzymatic reaction was accelerated by microwave-assisted heating (2450 MHz, power of 100 W) at 38°C. The enzymatic reaction lasted for 2 h, and stirring was maintained during the reaction to promote the release of protein.

[0074] (4) Eggshell peptide purification and freeze-drying: After the enzymatic hydrolysis reaction is completed, the supernatant is obtained by centrifugation at 4000 rpm for 16 min. The supernatant is concentrated and purified by an ultrafiltration device, and an ultrafiltration membrane with a molecular weight cutoff of 10 kDa is used to remove small molecular impurities and residual enzymes to obtain a high-purity protein solution. The purified protein solution is vacuum freeze-dried to obtain a solid protein powder, i.e., eggshell peptide, with a protein purity of 73%.

[0075] The extracted eggshell peptide can be used for the preparation of facial cream, and the components of the facial cream include component 1 (water phase component), component 2 (oil phase component) and component 3 (component added in the cooling stage).

[0076] Component 1 (aqueous phase): 170 g distilled water, 4.5 g glycerol, 2.5 g colloidal oatmeal;

[0077] Component 2 (oil phase ingredients): avocado butter 7g, grape seed oil 20g, emulsifying wax 11g;

[0078] Component 3 (ingredients added during the cooling stage): 5.5 g of extracted eggshell peptide, 2.2 g of phenoxyethanol, 2.2 g of vitamin E, and 1.2 g of vanilla essential oil.

[0079] Preparation of the cream: Heat the water phase and oil phase ingredients separately at 75°C in a double boiler until they are completely melted. Mix the two phases and stir them at 3000 rpm for 4 minutes to form an emulsion. After the emulsion cools, add the ingredients from the cooling stage and mix well. Store the finished cream in a dark container to prevent the active ingredients from being destroyed by light.

[0080] The above embodiment is only one of the preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A method for extracting eggshell peptides based on enzyme immobilization technology, characterized in that: include: S1. Crush the egg shell with membrane; S2, preparing immobilized enzyme: dissolving neutral protease in phosphate buffer, mixing with inert carrier solution, stirring to obtain a mixed solution; dropping the mixed solution into CaCl2 solution, stirring to obtain a small spherical product, filtering and washing, adding the small spherical product into glutaraldehyde solution for cross-linking reaction, filtering, washing and drying after the reaction to obtain neutral protease, i.e., immobilized enzyme; S3, enzymatic extraction: suspend the crushed eggshell in phosphate buffer, add immobilized enzyme at a mass ratio of 0.8-1.2:10 of enzyme to eggshell, perform enzymatic hydrolysis for 1.8-2.2h at 35-38℃, and stir continuously during the enzymatic hydrolysis; S4. After the enzymatic hydrolysis reaction is completed, the supernatant is obtained by centrifugation, and the supernatant is concentrated and purified by an ultrafiltration device to obtain a protein solution, which is then freeze-dried in vacuum to obtain a solid protein powder, i.e., eggshell peptide.

2. The eggshell peptide extraction method based on enzyme immobilization technology according to claim 1, characterized in that: In step S1, the egg shell with membrane is crushed to 50-100 μm.

3. The eggshell peptide extraction method based on enzyme immobilization technology according to claim 2 is characterized in that: In step S2, 0.4-0.6 g of neutral protease is dissolved in 100 mL of phosphate buffer with a pH of 7.2-7.8, and then mixed with an inert carrier solution at a mass ratio of 1:5, and stirred for 0.8-1.2 hours to obtain a mixed solution; the mixed solution is dropped into 50-60 mL of CaCl2 solution from a height of 8-12 cm, stirred at 24-26° C. to obtain a small spherical product, which is filtered and washed with distilled water; the small spherical product is added to 40-60 mL of glutaraldehyde solution, cross-linked at 35-38° C. for 1.5-2.5 hours, and the immobilized enzyme is obtained after filtering, washing and low-temperature drying.

4. The eggshell peptide extraction method based on enzyme immobilization technology according to claim 3 is characterized in that: The inert carrier solution is agarose microsphere or silica microsphere solution with a mass concentration of 4%; the mass concentration of the CaCl2 solution is 2%; and the mass concentration of the glutaraldehyde solution is 0.5%.

5. The eggshell peptide extraction method based on enzyme immobilization technology according to any one of claims 1, 3 or 4, characterized in that: In step S3, the pH value of the phosphate buffer is 7.2-7.8, and the crushed egg shells are suspended in the phosphate buffer at a concentration of 95-105 g / L.

6. The eggshell peptide extraction method based on enzyme immobilization technology according to claim 5, characterized in that: In step S3, after each enzymatic hydrolysis reaction is completed, the immobilized enzyme is separated from the solution by filtration or centrifugation, and the immobilized enzyme can be recycled for 5-10 times.

7. The eggshell peptide extraction method based on enzyme immobilization technology according to claim 6, characterized in that: In step S4, the centrifugation conditions are: 3800-4000 rpm for 12-16 min; the ultrafiltration membrane in the ultrafiltration device has a molecular weight cutoff of 10 kDa.

8. Use of the eggshell peptide extracted by the extraction method according to any one of claims 1 to 7 in facial cream.

9. The use of the eggshell peptide according to claim 8, characterized in that: The components of facial cream include water phase ingredients, oil phase ingredients and ingredients added during the cooling stage; Water phase ingredients: 150-170 parts of distilled water, 3.5-4.5 parts of glycerin, 1.5-2.5 parts of colloidal oatmeal; Oil phase ingredients: 5-7 parts avocado butter, 16-20 parts grape seed oil, 9-11 parts emulsifying wax; Ingredients added during the cooling stage: 4.5-5.5 parts of extracted eggshell peptides, 1.8-2.2 parts of phenoxyethanol, 1.8-2.2 parts of vitamin E, and 0.8-1.2 parts of vanilla essential oil.

10. The use of the eggshell peptide according to claim 9, characterized in that: The preparation method of the facial cream is as follows: heating the water phase component and the oil phase component at a temperature of 70-80° C. respectively until they are completely melted, mixing the two phases, stirring with a stirrer at 3000-3500 rpm for 3-5 minutes to form an emulsion, and after the emulsion is cooled, adding the cooling stage added components and mixing well to obtain the facial cream.

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