An egg yolk-derived active peptide for promoting bone growth and a preparation method thereof

Abstract

The application relates to the technical field of active peptides, and discloses an egg yolk-derived active peptide for promoting bone growth and a preparation method. The preparation method is as follows: the poultry eggs are cleaned, selected, sterilized and dried to obtain sterile poultry eggs; the egg yolk is subjected to spray drying to obtain egg yolk powder, and the egg yolk powder is subjected to defatting treatment to obtain defatted egg yolk powder; the defatted egg yolk powder is sieved, and deionized water is added to obtain a defatted egg yolk powder suspension; the pH and temperature of the defatted egg yolk powder suspension are adjusted, and enzyme hydrolysis is carried out to obtain an enzyme hydrolysate; the enzyme hydrolysate is inactivated and stored; the enzyme hydrolysate is added with calcium lactate and zinc lactate, and thermophilic fat Bacillus stearothermophilus is added, and fermentation is carried out to obtain a fermentation liquor; and an active peptide solution is separated. The application adopts the above egg yolk-derived active peptide for promoting bone growth and the preparation method, small-molecular-weight active peptides are obtained through step-by-step enzyme hydrolysis and fermentation, the active peptides are easy to absorb, and calcium ions and zinc ions are added during the fermentation, so that the active peptides are chelated with metal ions, growth and bone development are promoted.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to a bioactive peptide derived from egg yolk that promotes bone growth and its preparation method. Background Technology

[0002] Poultry eggs, as one of the most basic foods in people's daily diet, are rich in protein, lipids, minerals, and vitamins. Studies have reported that the protein in poultry eggs contains 18 essential amino acids, which are easily digested and absorbed by the human body, making them extremely valuable biologically. Egg yolks, being the most nutritious part of poultry eggs, also possess strong emulsifying and gelling properties, making them a food ingredient with high application value. Defatted egg yolk protein, a byproduct of egg yolk lecithin extraction, has a balanced amino acid profile and is a high-quality protein. Egg yolk protein is rich in highly bioactive polypeptide fragments, possessing high added value. Furthermore, after protease hydrolysis, its allergenicity decreases, its digestibility increases, and its bioactivity becomes even richer. Therefore, it is often used to prepare bioactive peptides, achieving the modification and utilization of byproducts.

[0003] Osteoporosis is a chronic skeletal disease characterized by decreased bone mass and destruction of bone microstructure, primarily affecting the elderly and postmenopausal women. With the increasing aging of the population, developing safe and effective prevention and treatment methods is extremely important. Poultry eggs (especially egg yolks) are rich in functional proteins, such as vitellin and globulin, which can release bioactive peptides through enzymatic hydrolysis and fermentation. Egg yolk peptides show potential in the field of bone health due to their good absorption and bioactivity. Therefore, this invention provides an active peptide derived from egg yolk that promotes bone growth, thereby addressing the problem of osteoporosis. Summary of the Invention

[0004] The purpose of this invention is to provide an active peptide derived from egg yolk that promotes bone growth and its preparation method. The active peptide is obtained by stepwise enzymatic hydrolysis and fermentation, which makes it easy to absorb. At the same time, calcium ions and zinc ions are added during fermentation to allow the active peptide to chelate metal ions, thereby promoting growth and bone development.

[0005] To achieve the above objectives, the present invention provides a method for preparing an active peptide derived from egg yolk that promotes bone growth, comprising the following steps:

[0006] Step 1: Clean the poultry eggs, then select them by light to remove spoiled eggs, sterilize and dry them to obtain sterile poultry eggs;

[0007] Step 2: Crush the eggs, remove the yolks and set them aside. Spray dry the yolks to obtain yolk powder. Defatt the yolk powder to obtain defatted yolk powder.

[0008] Step 3: Pass the defatted egg yolk powder through a 40-mesh sieve, add deionized water to dissolve the defatted egg yolk powder, and then sterilize at 62-65℃ for 30 minutes to obtain a defatted egg yolk powder suspension. Cool to room temperature for later use.

[0009] Step 4: Adjust the pH and temperature of the defatted egg yolk powder suspension, and perform stepwise enzymatic hydrolysis to obtain the hydrolysate;

[0010] Step 5: Heat the enzyme hydrolysate to 65-90℃, inactivate for 30 minutes, and cool before use;

[0011] Step 6: Add calcium lactate and zinc lactate to the cooled enzymatic hydrolysate, and add Bacillus stearothermophilus. Ferment at 50-55℃ for 3-4 hours, and obtain the fermentation broth after cooling.

[0012] Step 7: The fermentation broth is separated using an 800-1500 Da membrane to obtain an active peptide solution.

[0013] Furthermore, in step 1, the poultry eggs are one or more of the following: chicken eggs, duck eggs, goose eggs, ostrich eggs, or quail eggs.

[0014] Furthermore, in step 3, the solid-liquid ratio of defatted egg yolk powder to deionized water is 1g:15-20mL.

[0015] Furthermore, the specific steps of enzymatic hydrolysis in step 4 are as follows:

[0016] ① Add alkaline protease and hydrolyze for 2-3 hours at 40-45℃ and pH 9.0-11.0. The amount of alkaline protease added is 1.2%.

[0017] ② Add neutral protease and hydrolyze for 2-3 hours at 45-50℃ and pH 5.5-8.5. The amount of neutral protease added is 1.0%.

[0018] ③ Add flavor protease and hydrolyze for 2-3 hours at 50-55℃ and pH 6.5-7.5. The amount of flavor protease added is 0.8%.

[0019] Furthermore, in step 6, the amount of calcium lactate added is 30-40 mg / kg, the amount of zinc lactate added is 30-50 mg / kg, and the thermophilic Bacillus stearothermophilus is Bacillus stearothermophilus CCTCC AB 2013010, with an inoculation amount of 3%.

[0020] Furthermore, in step 7, a 1000-1200 Da membrane is used for separation to obtain an active peptide solution.

[0021] The present invention also provides an active peptide derived from egg yolk that promotes bone growth, wherein the active peptide is prepared by the above-described preparation method.

[0022] Furthermore, the molecular weight of the active peptide is 1000-1200 Da.

[0023] This invention also provides the application of the above-mentioned active peptide derived from egg yolk that promotes bone growth in the preparation of products that promote bone growth.

[0024] Furthermore, the product includes pharmaceuticals.

[0025] The advantages and positive effects of the egg yolk-derived bioactive peptide for promoting bone growth and its preparation method described in this invention are as follows:

[0026] 1. This invention prepares small molecular weight active peptides by first performing stepwise enzymatic hydrolysis and then high-temperature fermentation, which are easily absorbed. At the same time, calcium and zinc ions are added during fermentation to enable the active peptides to chelate metal ions, thereby promoting growth and bone development.

[0027] 2. During the high-temperature fermentation process in this invention, calcium and zinc ions are added, which allows the small molecular weight peptides to chelate with calcium and zinc ions. This not only enhances bone calcium content and bone density but also promotes calcium absorption and bone growth.

[0028] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0029] The technical solution of the present invention will be further described below through embodiments.

[0030] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0031] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0032] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0033] Example 1

[0034] A method for preparing an active peptide derived from egg yolk that promotes bone growth includes the following steps:

[0035] Step 1: Wash the goose eggs, then select them by light to remove spoiled goose eggs, sterilize and dry them to obtain sterile goose eggs;

[0036] Step 2: Crush the goose eggs, remove the yolks and set them aside. Spray dry the yolks to obtain yolk powder. Then defatt the yolk powder to obtain defatted yolk powder.

[0037] Step 3: Pass the defatted egg yolk powder through a 40-mesh sieve, add deionized water, and the solid-liquid ratio of defatted egg yolk powder to deionized water is 1g:18mL to dissolve the defatted egg yolk powder. Then sterilize at 63℃ for 30min to obtain a defatted egg yolk powder suspension, and cool to room temperature for later use.

[0038] Step 4: Adjust the pH and temperature of the defatted egg yolk powder suspension, and perform stepwise enzymatic hydrolysis to obtain the hydrolysate;

[0039] The specific steps are as follows:

[0040] ① Add alkaline protease and hydrolyze for 2 hours at 40-45℃ and pH 9.0-11.0. The amount of alkaline protease added is 1.2%.

[0041] ② Add neutral protease and hydrolyze for 2.5 hours at 45-50℃ and pH 5.5-8.5. The amount of neutral protease added is 1.0%.

[0042] ③ Add flavor protease and hydrolyze for 2 hours at 50-55℃ and pH 6.5-7.5. The amount of flavor protease added is 0.8%.

[0043] Step 5: Heat the enzyme hydrolysate to 75°C, inactivate it for 30 minutes, and then cool it for later use.

[0044] Step 6: Add 35 mg / kg calcium lactate and 30 mg / kg zinc lactate to the cooled enzymatic hydrolysate, and add 3% of Bacillus stearothermophilus CCTCC AB 2013010. Ferment at 50-55℃ for 3 hours, and obtain the fermentation broth after cooling.

[0045] Step 7: The fermentation broth is separated using a 1000 Da membrane to obtain an active peptide solution.

[0046] Example 2

[0047] A method for preparing an active peptide derived from egg yolk that promotes bone growth includes the following steps:

[0048] Step 1: Wash the eggs, then select them by light to remove spoiled eggs, sterilize and dry them to obtain sterile eggs;

[0049] Step 2: Crush the eggs, remove the yolks and set them aside. Spray dry the yolks to obtain yolk powder. Then defatt the yolk powder to obtain defatted yolk powder.

[0050] Step 3: Pass the defatted egg yolk powder through a 40-mesh sieve, add deionized water, and the solid-liquid ratio of defatted egg yolk powder to deionized water is 1g:20mL to dissolve the defatted egg yolk powder. Then sterilize at 63℃ for 30min to obtain a defatted egg yolk powder suspension, and cool to room temperature for later use.

[0051] Step 4: Adjust the pH and temperature of the defatted egg yolk powder suspension, and perform stepwise enzymatic hydrolysis to obtain the hydrolysate;

[0052] The specific steps are as follows:

[0053] ① Add alkaline protease and hydrolyze for 2 hours at 40-45℃ and pH 9.0-11.0. The amount of alkaline protease added is 1.2%.

[0054] ② Add neutral protease and hydrolyze for 2.5 hours at 45-50℃ and pH 5.5-8.5. The amount of neutral protease added is 1.0%.

[0055] ③ Add flavor protease and hydrolyze for 2 hours at 50-55℃ and pH 6.5-7.5. The amount of flavor protease added is 0.8%.

[0056] Step 5: Heat the enzyme hydrolysate to 85°C, inactivate it for 30 minutes, and then cool it for later use.

[0057] Step 6: Add 40 mg / kg calcium lactate and 40 mg / kg zinc lactate to the cooled enzymatic hydrolysate, and add 3% of Bacillus stearothermophilus CCTCC AB 2013010. Ferment at 50-55℃ for 3.5 h, and obtain the fermentation broth after cooling.

[0058] Step 7: The fermentation broth is separated using a 1000 Da membrane to obtain an active peptide solution.

[0059] Example 3

[0060] A method for preparing an active peptide derived from egg yolk that promotes bone growth includes the following steps:

[0061] Step 1: Wash the duck eggs, then select them by light to remove spoiled duck eggs, sterilize and dry them to obtain sterile duck eggs;

[0062] Step 2: Crush the duck eggs, remove the yolks for later use, spray dry the yolks to obtain yolk powder, and defatt the yolk powder to obtain defatted yolk powder;

[0063] Step 3: Pass the defatted egg yolk powder through a 40-mesh sieve, add deionized water, and the solid-liquid ratio of defatted egg yolk powder to deionized water is 1g:15mL to dissolve the defatted egg yolk powder. Then sterilize at 63℃ for 30min to obtain a defatted egg yolk powder suspension, and cool to room temperature for later use.

[0064] Step 4: Adjust the pH and temperature of the defatted egg yolk powder suspension, and perform stepwise enzymatic hydrolysis to obtain the hydrolysate;

[0065] The specific steps are as follows:

[0066] ① Add alkaline protease and hydrolyze for 2 hours at 40-45℃ and pH 9.0-11.0. The amount of alkaline protease added is 1.2%.

[0067] ② Add neutral protease and hydrolyze for 2.5 hours at 45-50℃ and pH 5.5-8.5. The amount of neutral protease added is 1.0%.

[0068] ③ Add flavor protease and hydrolyze for 2 hours at 50-55℃ and pH 6.5-7.5. The amount of flavor protease added is 0.8%.

[0069] Step 5: Heat the enzyme hydrolysate to 80°C, inactivate it for 30 minutes, and then cool it for later use.

[0070] Step 6: Add 30 mg / kg calcium lactate and 50 mg / kg zinc lactate to the cooled enzymatic hydrolysate, and add 3% of Bacillus stearothermophilus CCTCC AB 2013010. Ferment at 50-55℃ for 3.5 h, and obtain the fermentation broth after cooling.

[0071] Step 7: The fermentation broth is separated using a 1000 Da membrane to obtain an active peptide solution.

[0072] Comparative Example 1

[0073] A method for preparing an active peptide derived from egg yolk that promotes bone growth includes the following steps:

[0074] Step 1: Wash the goose eggs, then select them by light to remove spoiled goose eggs, sterilize and dry them to obtain sterile goose eggs;

[0075] Step 2: Crush the goose eggs, remove the yolks and set them aside. Spray dry the yolks to obtain yolk powder. Then defatt the yolk powder to obtain defatted yolk powder.

[0076] Step 3: Pass the defatted egg yolk powder through a 40-mesh sieve, add deionized water, and the solid-liquid ratio of defatted egg yolk powder to deionized water is 1g:18mL to dissolve the defatted egg yolk powder. Then sterilize at 63℃ for 30min to obtain a defatted egg yolk powder suspension, and cool to room temperature for later use.

[0077] Step 4: Add 35 mg / kg calcium lactate and 30 mg / kg zinc lactate to the defatted egg yolk powder suspension, and add 3% of thermophilic Bacillus stearothermophilus CCTCC AB 2013010. Ferment at 50-55℃ for 3 hours, and obtain the fermentation broth after cooling.

[0078] Step 5: Adjust the pH and temperature of the fermentation broth, and perform stepwise enzymatic hydrolysis to obtain the enzymatic hydrolysate;

[0079] The specific steps are as follows:

[0080] ① Add alkaline protease and hydrolyze for 2 hours at 40-45℃ and pH 9.0-11.0. The amount of alkaline protease added is 1.2%.

[0081] ② Add neutral protease and hydrolyze for 2.5 hours at 45-50℃ and pH 5.5-8.5. The amount of neutral protease added is 1.0%.

[0082] ③ Add flavor protease and hydrolyze for 2 hours at 50-55℃ and pH 6.5-7.5. The amount of flavor protease added is 0.8%.

[0083] Step 6: Heat the enzyme hydrolysate to 75°C, inactivate it for 30 minutes, and then cool it for later use.

[0084] Step 7: After cooling, the enzymatic hydrolysate is separated using a 1000 Da membrane to obtain an active peptide solution.

[0085] Comparative Example 2

[0086] A method for preparing an active peptide derived from egg yolk that promotes bone growth includes the following steps:

[0087] Step 1: Wash the goose eggs, then select them by light to remove spoiled goose eggs, sterilize and dry them to obtain sterile goose eggs;

[0088] Step 2: Crush the goose eggs, remove the yolks and set them aside. Spray dry the yolks to obtain yolk powder. Then defatt the yolk powder to obtain defatted yolk powder.

[0089] Step 3: Pass the defatted egg yolk powder through a 40-mesh sieve, add deionized water, and the solid-liquid ratio of defatted egg yolk powder to deionized water is 1g:18mL to dissolve the defatted egg yolk powder. Then sterilize at 63℃ for 30min to obtain a defatted egg yolk powder suspension, and cool to room temperature for later use.

[0090] Step 4: Adjust the pH and temperature of the defatted egg yolk powder suspension, and perform stepwise enzymatic hydrolysis to obtain the hydrolysate;

[0091] The specific steps are as follows:

[0092] ① Add alkaline protease and hydrolyze for 2 hours at 40-45℃ and pH 9.0-11.0. The amount of alkaline protease added is 1.2%.

[0093] ② Add neutral protease and hydrolyze for 2.5 hours at 45-50℃ and pH 5.5-8.5. The amount of neutral protease added is 1.0%.

[0094] ③ Add flavor protease and hydrolyze for 2 hours at 50-55℃ and pH 6.5-7.5. The amount of flavor protease added is 0.8%.

[0095] Step 5: Heat the enzyme hydrolysate to 75°C, inactivate it for 30 minutes, and then cool it for later use.

[0096] Step 6: Add 35 mg / kg calcium lactate and 30 mg / kg zinc lactate to the cooled enzymatic hydrolysate, and add 3% Bacillus subtilis CCTCC AB 130001. Ferment at 28-30℃ for 3 hours, and obtain the fermentation broth after cooling.

[0097] Step 7: The fermentation broth is separated using a 1000 Da membrane to obtain an active peptide solution.

[0098] The results showed

[0099] The active peptide solutions prepared in Examples 1-3 and Comparative Examples 1-2 were used in rat experiments to verify their effects.

[0100] ① Bone mineral density test:

[0101] Female SD rats, weighing approximately 300 grams, were acclimatized for one week before the experiment. Ovarian removal rat model was established: Rats were anesthetized by intraperitoneal injection of sodium pentobarbital solution (30 mg / kg BW). After fixation in the abdominal position, hair was removed 3-4 cm from the vaginal opening along the midline of the abdomen. The area was disinfected with iodine and alcohol, and after slight drying, the skin and abdominal muscles were incised approximately 2-3 cm deep. White fat was visible through the incision. After separating the fat layer to locate the uterus, one uterine horn was gently pulled out, revealing the ovary encased in a fat mass at its end. The fat mass was separated, revealing a pink or yellowish-red ovary. The ovary was clamped with hemostatic forceps, and the fallopian tube (including fat) below the ovary was ligated with silk suture. The ovary was then excised (checking for complete removal), and the uterine horn was returned to the abdominal cavity. The other ovary was excised in the same manner. The abdominal muscles and skin were sutured in layers and disinfected again. Finally, 20,000 U of penicillin was injected intramuscularly into the hind limb. Five days after ovariectomy, vaginal smears were examined in rats (a small amount of physiological saline was drawn with a dropper, gently inserted into the vagina 1-2 cm, rinsed several times, aspirated, and smeared on a glass slide for observation under a microscope). This was done once a day for 7 consecutive days to check whether the rat ovariectomy was complete, thus successfully establishing a rat model of ovariectomy.

[0102] Experimental grouping: Rats were divided into 6 groups, including 5 experimental groups and 1 control group, with 8 rats in each group. Each experimental group was fed 10 mL of the active peptide solution of Examples 1, 2, 3 or Comparative Examples 1, 2, and 3, respectively, while the control group was fed 10 mL of distilled water. All other conditions were the same. Feeding times were 9:00 AM and 3:00 PM daily for 3 months, with the same diet used throughout the experiment.

[0103] Bone calcium determination: The left femur of a rat was dried in an oven at 105℃ to constant weight, and the dry weight was measured. The bone was then digested in an Erlenmeyer flask, and the bone calcium content was determined by atomic absorption spectrometry. Bone mineral density determination: The right femur was used, and the bone mineral density at the midpoint and distal end of the femur was measured using a bone densitometer. The results are shown in Table 1.

[0104] Table 1

[0105]

[0106] As shown in Table 1, compared with the control group, the bone calcium content, femoral midpoint and distal femoral bone mineral density of rats in Examples 1, 2 and 3 were significantly higher, indicating that the active peptides prepared in Examples 1, 2 and 3 can promote the increase of femoral bone mineral density, thereby promoting bone growth.

[0107] The order of fermentation and enzymatic hydrolysis in Comparative Example 1 is different from that in Example 1. The bone calcium content and bone density of rats in Comparative Example 1 are lower than those in Example 1, indicating that enzymatic hydrolysis followed by fermentation can produce bioactive peptides with smaller molecular weights, thereby increasing bone calcium content and bone density and promoting bone growth.

[0108] Compared with Example 1, the fermentation temperature in Comparative Example 2 was lower, and the bone calcium content and bone density of rats in Comparative Example 2 were lower than those in Example 1. This indicates that fermentation with Bacillus stearothermophilus at a higher temperature allows peptides to chelate with metal ions (calcium ions and zinc ions), while producing active peptides with smaller molecular weights, thereby increasing bone calcium content and bone density in rats and promoting bone growth.

[0109] ② Bone calcium absorption:

[0110] Weaned rats approximately 4 weeks old were separated into different cages and housed for 4 weeks after a 1-week acclimatization period. The rats were divided into 6 groups: 5 experimental groups and 1 control group, with 8 rats in each group. Each experimental group was fed 5 mL of the active peptide solution from Examples 1, 2, and 3, or Comparative Examples 1 and 2, respectively, while the control group was fed 5 mL of distilled water. All other conditions were the same. Deionized water was provided during the experiment to avoid calcium acquisition from drinking water. A 3-day calcium metabolism experiment was conducted after 3 weeks of experimentation. Food intake was recorded over 3 days, and fecal samples were collected over 72 hours to determine the calcium content in the feed and feces.

[0111] Calcium intake (mg / d) = Calcium content in feed (mg / g) × Feed consumption (g / d);

[0112] Fecal calcium (mg / d) = Calcium content in feces (mg / g) × Fecal excretion (g / d);

[0113] Apparent calcium absorption rate (%) = (calcium intake - fecal calcium) / calcium intake × 100%;

[0114] The results are shown in Table 2:

[0115] Table 2

[0116]

[0117] As shown in Table 2, compared with the control group, the bone calcium absorption rate of rats in Examples 1, 2, and 3 was higher, indicating that the active peptides prepared in Examples 1, 2, and 3 can promote bone calcium absorption, thereby promoting bone growth.

[0118] The order of fermentation and enzymatic hydrolysis in Comparative Example 1 is different from that in Example 1. The bone calcium absorption rate of rats in Comparative Example 1 is lower than that in Example 1, indicating that enzymatic hydrolysis followed by fermentation can produce active peptides with smaller molecular weights, thereby increasing the bone calcium absorption rate and promoting bone growth.

[0119] Compared with Example 1, the fermentation temperature in Comparative Example 2 was lower, and the bone calcium absorption rate of rats in Comparative Example 2 was lower than that in Example 1. This indicates that fermentation with Bacillus stearothermophilus at a higher temperature allows peptides to chelate with metal ions (calcium ions and zinc ions), while producing active peptides with smaller molecular weights, thereby increasing the bone calcium absorption rate of rats and promoting bone growth.

[0120] Therefore, the present invention adopts the above-mentioned active peptide for promoting bone growth derived from egg yolk and its preparation method. The active peptide with small molecular weight is obtained by stepwise enzymatic hydrolysis and fermentation, which is easy to absorb. At the same time, calcium ions and zinc ions are added during fermentation, so that the active peptide chelates metal ions and promotes growth and bone development.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing an active peptide derived from egg yolk that promotes bone growth, characterized in that, Includes the following steps: Step 1: Clean the poultry eggs, then select them by light to remove spoiled eggs, sterilize and dry them to obtain sterile poultry eggs; Step 2: Crush the eggs, remove the yolks and set them aside. Spray dry the yolks to obtain yolk powder. Defatt the yolk powder to obtain defatted yolk powder. Step 3: Pass the defatted egg yolk powder through a 40-mesh sieve, add deionized water to dissolve the defatted egg yolk powder, and then sterilize at 62-65℃ for 30 minutes to obtain a defatted egg yolk powder suspension. Cool to room temperature for later use. Step 4: Adjust the pH and temperature of the defatted egg yolk powder suspension, and perform stepwise enzymatic hydrolysis to obtain the hydrolysate; The specific steps for stepwise enzymatic hydrolysis are as follows: ① Add alkaline protease and hydrolyze for 2-3 hours at 40-45℃ and pH 9.0-11.

0. The amount of alkaline protease added is 1.2%. ② Add neutral protease and hydrolyze for 2-3 hours at 45-50℃ and pH 5.5-8.

5. The amount of neutral protease added is 1.0%. ③ Add flavor protease and enzymatically hydrolyze for 2-3 hours at 50-55℃ and pH 6.5-7.

5. The amount of flavor protease added is 0.8%. Step 5: Heat the enzyme hydrolysate to 65-90℃, inactivate for 30 minutes, and cool before use; Step 6: Add calcium lactate and zinc lactate to the cooled enzymatic hydrolysate, and add Bacillus stearothermophilus. Ferment at 50-55℃ for 3-4 hours, and obtain the fermentation broth after cooling. The thermophilic Bacillus stearothermophilus was Bacillus stearothermophilus CCTCCAB 2013010, and the inoculum size was 3%. Step 7: The fermentation broth is separated using a 1000 Da membrane to obtain an active peptide solution.

2. The method for preparing an active peptide derived from egg yolk that promotes bone growth according to claim 1, characterized in that: In step 1, the poultry eggs are one or more of the following: chicken eggs, duck eggs, goose eggs, ostrich eggs, or quail eggs.

3. The method for preparing an active peptide derived from egg yolk that promotes bone growth according to claim 1, characterized in that: In step 3, the solid-liquid ratio of defatted egg yolk powder to deionized water is 1g:15-20mL.

4. The method for preparing an active peptide derived from egg yolk that promotes bone growth according to claim 1, characterized in that: In step 6, the amount of calcium lactate added is 30-40 mg / kg, and the amount of zinc lactate added is 30-50 mg / kg.

5. An active peptide derived from egg yolk that promotes bone growth, characterized in that: The active peptide was prepared using the preparation method described in any one of claims 1-4.

6. The use of the egg yolk-derived active peptide for promoting bone growth as described in claim 5 in the preparation of a drug for promoting bone growth.

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