Yolk protein osteogenic peptide and preparation method thereof

Abstract

The application discloses egg yolk protein osteogenic peptides and a preparation method thereof, and belongs to the fields of biological medicines, functional food or egg product processing technologies. The application adopts defatted egg yolk powder enzymolysis to prepare osteogenic active peptides, and the osteogenic active peptides are enriched and separated through macroporous resins; the preparation and enrichment process is suitable for large-scale production, and can promote transformation from laboratory achievements to industrial production; two osteogenic active peptides are separated and identified, wherein, DFDLPT has the function of promoting osteoblast proliferation activity, and FDIDPG has the function of promoting osteoblast differentiation and mineralization.

Description

Technical Field

[0001] This invention relates to an egg yolk protein osteogenic peptide and its preparation method, belonging to the fields of biomedicine, functional food or egg product processing technology. Background Technology

[0002] Egg protein and its hydrolysates have been proven to possess various biological activities, such as anti-inflammatory, antioxidant, and anti-cancer properties, making them excellent raw materials for developing bioactive peptide functional foods. In the prevention and treatment of osteoporosis, various proteins in egg yolks have shown positive regulatory effects on bone growth and development, as well as the proliferation, differentiation, and expression of osteocytes in multiple in vivo and in vitro experiments. The main component of defatted egg yolk powder is egg yolk protein, which has the potential to be developed into bioactive peptides.

[0003] Human bone metabolism is a complex process, a dynamic balance between osteoclasts resorbing old bone and osteoblasts forming new bone. With age, bone regeneration capacity weakens. When the resorption of old bone exceeds the formation of new bone, net bone loss occurs, leading to osteoporosis, characterized by low bone density, altered bone microstructure, and increased fracture risk. Osteoporosis is a typical disease induced by the disruption of skeletal homeostasis, commonly seen in middle-aged and elderly individuals, especially postmenopausal women with primary osteoporosis due to estrogen deficiency. Calcium and vitamin D (VD) supplementation has become a common method to improve bone density. However, recent studies have increasingly found that many people do not achieve the expected results from calcium and VD supplementation. Furthermore, excessive calcium intake can lead to side effects such as kidney stones and vascular calcification. Therefore, developing safe and novel bone-strengthening functional factors has become a research hotspot in the field of bone metabolism. Egg yolk peptides, as food-derived bioactive peptides, have great potential to promote bone health in the food industry.

[0004] Significant progress has been made in recent years in the study of osteogenic activity of egg yolk peptides. Current research mainly focuses on two types of bioactive peptides: (1) phosphorylated peptides that improve calcium homeostasis through calcium ion chelation; and (2) signal peptides that directly regulate osteoblast proliferation and differentiation pathways. The former can be traced back to the pioneering work of Choi's team in 2005, where the phosphopeptides isolated from egg yolk high phosphoprotein not only significantly increased the mineral density of rat bones, but also revealed the molecular mechanism by which egg yolk peptides enhance calcium bioavailability by inhibiting the formation of calcium phosphate precipitation. In the past decade, Professor Ma Meihu's research group at Huazhong Agricultural University has completed several studies on the effects of egg yolk high phosphoprotein on the proliferation, differentiation, mineralization and other functions of osteoblast MC3T3-E1, as well as its influence on chicken embryo bone development, and has conducted in-depth research on the osteogenic function of egg yolk high phosphoprotein. Research at the University of Alberta in Canada found that compared with the intact egg yolk high phosphoprotein, the peptides obtained by its hydrolysis showed a higher ability to promote osteogenic differentiation and biomineralization in osteoblasts, and proposed the hypothesis that the degree of phosphorylation may be proportional to the bone regulatory bioactivity.

[0005] Subsequent studies have shown that the calcium-binding capacity of these peptides is closely related to the phosphate group content and molecular weight. However, the preparation, separation, and purification of key active ingredients rely on complex processes (such as protein purification and chromatographic chromatography), which are costly, inefficient, and difficult to adapt to the needs of industrial continuous production. The second type of research targeting osteoblast regulation began with the discovery at Semyung University in South Korea in 2008: the water-soluble peptide YPEP obtained by enzymatic hydrolysis of egg yolk protein can activate the expression of the Runx2 gene and bidirectionally regulate osteoblast differentiation (promoting mineralization) and osteoclast activity (inhibiting bone resorption), thus elucidating the anti-osteoporosis potential of egg yolk peptides at the molecular level for the first time.

[0006] Therefore, screening for osteogenic peptides from egg yolk protein and developing a method for preparing compositions containing osteogenic peptides has extremely high practical and economic value. Summary of the Invention

[0007] To address the aforementioned issues, this invention prepares an egg yolk protein hydrolysate with osteogenic activity by enzymatically degreasing egg yolk powder. Furthermore, it further separates and purifies the hydrolysate to prepare an egg yolk protein peptide composition with even stronger osteogenic activity, as well as egg yolk protein peptides FDIDPG and DFDLPT.

[0008] The first objective of this invention is to provide an egg yolk protein peptide with osteogenic activity, wherein the egg yolk protein peptide is FDIDPG or DFDLPT, and the amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0009] A second objective of this invention is to provide a method for preparing egg yolk protein hydrolysate, wherein the egg yolk protein hydrolysate contains the aforementioned egg yolk protein peptides, comprising the following steps:

[0010] (1) Mix defatted egg yolk powder with water, sonicate to obtain a turbid liquid; adjust the pH of the turbid liquid to 10~11, add alkaline protease with E / S=0.5~1.5%, and enzymatically hydrolyze for 1.0~1.5 h to obtain a first enzymatic hydrolysate;

[0011] (2) Adjust the pH of the first enzymatic hydrolysate to 6.5-7, add a complex protease with E / S = 0.8-1.5%, and enzymatically hydrolyze for 2-8 h to obtain the second enzymatic hydrolysate; adjust the pH of the second enzymatic hydrolysate to 6.5-7, inactivate the enzyme, centrifuge to collect the supernatant, and freeze-dry to obtain egg yolk protease hydrolysate.

[0012] In one embodiment, the ratio of defatted egg yolk powder to water in step (1) is 1 g: 7~15 mL;

[0013] Optionally, in step (1), the ultrasound is 100~300 W for 10~20 min.

[0014] In one embodiment, the defatted egg yolk powder in step (1) can be purchased supercritical CO2 defatted egg yolk powder.

[0015] In one embodiment, the alkaline protease in step (1) has an enzyme activity of 200,000 U / g, and the complex protease in step (2) is Protamex with an enzyme activity of 200,000 U / g.

[0016] A third object of the present invention is to provide egg yolk protein hydrolysates prepared by any of the methods described above.

[0017] A fourth object of the present invention is to provide a method for preparing an egg yolk protein peptide composition, the egg yolk protein peptide composition comprising the egg yolk protein peptide of claim 1, comprising the steps of:

[0018] (1) Mix defatted egg yolk powder with water, sonicate to obtain a turbid liquid; adjust the pH of the turbid liquid to 10~11, add alkaline protease with E / S=0.5~1.5%, and enzymatically hydrolyze for 1.0~1.5 h to obtain a first enzymatic hydrolysate;

[0019] (2) Adjust the pH of the first enzymatic hydrolysate to 6.5-7, add a complex protease with E / S = 0.8-1.5%, and enzymatically hydrolyze for 2-8 h to obtain the second enzymatic hydrolysate; adjust the pH of the second enzymatic hydrolysate to 6.5-7, inactivate the enzyme, sonicate, centrifuge to collect the supernatant, and freeze-dry to obtain egg yolk protease hydrolysate;

[0020] (3) Separate egg yolk protein hydrolysate using macroporous resin XA-1600, elute with ethanol aqueous solution with a volume fraction of 0~80%, collect the eluent, and dry to obtain a purified egg yolk protein peptide composition.

[0021] Optionally, in step (3), one column volume is eluted sequentially with deionized water, 20% ethanol aqueous solution, 40% ethanol aqueous solution, and 80% ethanol aqueous solution;

[0022] Optionally, the egg yolk protein peptide composition purified and separated in step (3) is obtained by elution and drying with a 20% (v / v) ethanol aqueous solution.

[0023] In one embodiment, the egg yolk protein peptide composition separated by the first purification can be further separated using a dextran gel Sephadex G-15, eluted with water, and the absorbance value is monitored at 220 nm to collect the egg yolk protein peptide composition separated by the second purification.

[0024] The elution flow rate was 1-2 mL / min, the elution time was 90-180 min, and the elution volume was 180 mL.

[0025] Optionally, the eluent obtained by collecting the first absorption peak on the elution curve is a yolk protein peptide composition separated by secondary purification;

[0026] Optionally, the elution period for the first absorption peak appearing on the elution curve is within the first 60 minutes.

[0027] In one embodiment, the egg yolk protein peptide composition separated by secondary purification can be further separated using dextran gel SP Spheadex C-25, eluted with a buffer containing 0~0.4M NaCl / acetic acid, and the absorbance value is monitored at 220 nm to collect the egg yolk protein peptide composition separated by tertiary purification.

[0028] Optionally, the elution flow rate is 1~1.5 mL / min, and the elution time for each eluent is 120~180 min;

[0029] Optionally, the eluent obtained by eluting with deionized water for 120 min is a yolk protein peptide composition purified and separated three times.

[0030] A fifth object of the present invention is to provide an egg yolk protein peptide composition prepared by any of the above methods.

[0031] The sixth object of the present invention is to provide a medicine or health product containing the above-mentioned egg yolk protein peptide or the above-mentioned egg yolk protein hydrolysate or the above-mentioned egg yolk protein peptide composition.

[0032] Optionally, the pharmaceuticals and health products may also contain derivatives of egg yolk protein peptides. These derivatives refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification, or glycosylation of the amino acid side chain groups, amino terminus, or carbonyl terminus of egg yolk protein peptides.

[0033] Optionally, the drug may further contain pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0034] Optionally, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0035] Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0036] Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0037] Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0038] Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers;

[0039] A seventh object of the present invention is to provide the use of the above-described egg yolk protein peptides, or the above-described egg yolk protein hydrolysates, or the above-described egg yolk protein peptide compositions, or any of the above-described methods, in the preparation of products that promote bone growth or improve bone density.

[0040] In one embodiment, the product includes orthopedic implant materials, bone repair scaffolds, calcium supplements, research reagents, drugs / therapeutic agents, and biomaterial complexes.

[0041] In one embodiment, the pharmaceutical or health product may also contain derivatives of egg yolk protein peptides. These derivatives refer to polypeptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification, or glycosylation of the amino acid side chain groups, amino terminus, or carbonyl terminus of egg yolk protein peptides.

[0042] Optionally, the drug may further contain pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0043] Optionally, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0044] Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0045] Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0046] Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0047] Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers;

[0048] Beneficial effects of the present invention

[0049] This invention uses defat egg yolk powder to enzymatically hydrolyze osteogenic active peptides, which are then enriched and separated using macroporous resin. The preparation and enrichment process is suitable for large-scale production and can promote the transformation of laboratory results into industrial production. This invention has isolated and identified two osteogenic active peptides, one of which has the activity of promoting osteoblast proliferation, and the other has the functional activity of promoting osteoblast differentiation and mineralization.

[0050] Specifically,

[0051] (1) The egg yolk protein hydrolysate EYP prepared in this invention does not inhibit the proliferation of osteoblast MC3T3-E1 cells in the concentration range of 25~200 μg / mL. After inducing MC3T3-E1 cells with 50 μg / mL EYP for 3, 4 and 5 days, ALP activity increased by 129.30%, 120.91% and 50.56%, respectively. Egg yolk protein hydrolysate EYP at concentrations of 25, 50 and 100 μg / mL promotes the expression of osteoblast differentiation-related genes and proteins and promotes cell mineralization.

[0052] (2) Furthermore, the osteogenic activity of the egg yolk protein peptide composition prepared by the present invention through separation and purification of egg yolk protein hydrolysate EYP is significantly improved compared with that of egg yolk protein hydrolysate EYP;

[0053] (3) Furthermore, the present invention identifies the egg yolk protein peptide composition and screens out the peptides DFDLPT and FDIDPG; wherein, the peptide DFDLPT has a significant proliferative effect on MC3T3-E1 cells, and the survival rate of MC3T3-E1 cells reaches 121.23% at a concentration of 100 μM; when the peptide FDIDPG is used to treat MC3T3-E1 cells at a concentration of 200 μM, the relative activity of ALP reaches 136.15%, the relative expression level of COL1A1 gene reaches 219.71%, and SPP1 reaches 295.44%; at the same time, the expression level of key osteoblast signaling proteins is significantly increased, and the cell mineralization level reaches the highest level. Attached Figure Description

[0054] Figure 1 The effect of enzymatic hydrolysis time on the yield of enzymatic hydrolysis products;

[0055] Figure 2 The effects of egg yolk protein hydrolysate concentration and induction time on ALP activity in osteoblast MC3T3-E1 cells;

[0056] Figure 3 The effect of egg yolk protein hydrolysate concentration on Runx2 protein expression in osteoblast MC3T3-E1 cells;

[0057] Figure 4 The effect of egg yolk protein hydrolysate concentration on the expression levels of COL1A1 and SPP1 genes in osteoblast MC3T3-E1 cells;

[0058] Figure 5 The effect of egg yolk protein hydrolysate concentration on osteoblast mineralization;

[0059] Figure 6 The results of separating egg yolk protein hydrolysates using macroporous resin XA-1600 are shown. In this figure, A is the chromatogram of separation by macroporous resin column; B is the ALP-promoting activity of the separated components EYP1, EYP2, EYP3, and EYP4; and C is the effect of each separated component on Runx2 protein expression.

[0060] Figure 7 The results of separating EYP2 using Sephadex G-15 dextran gel chromatography are shown. In the figure, A is the chromatogram of separation by Sephadex G-15 dextran gel chromatography column, B is the ALP-promoting activity of the separated components EYP2-1, EYP2-2, and EYP2-3, and C is the effect of each separated component on Runx2 protein expression.

[0061] Figure 8 The results of separating EYP2-1 using a dextran gel SP Spheadex C-25 column are shown. In the figure, A is the chromatogram of separation by the dextran gel Sephadex C-25 column, B is the ALP-promoting activity of the separated components EYP2-1-1 and EYP2-1-2, and C is the effect of each separated component on Runx2 protein expression.

[0062] Figure 9 The superimposed chromatograms show the molecular weight distributions of EYP, EYP2, EYP2-1, and EYP2-1-1.

[0063] Figure 10 The images show the total ion chromatogram of the EYP2-1-1 fraction and the primary mass spectra for retention times of 22.72–28.30 min and 33.22–38.79 min, respectively; where A is the total ion chromatogram, B is the primary mass spectrum for retention times of 22.72–28.30 min, and C is the primary mass spectrum for retention times of 33.22–38.79 min.

[0064] Figure 11 The images show the secondary mass spectra of the peptides, where A is peptide DSVSEPQEFS (peptide-1), B is ILPEDAPLD (peptide-2), C is DFDLPT (peptide-3), D is FDIDPG (peptide-4), and E is PLD (peptide-5).

[0065] Figure 12 The effects of peptides DSVSEPQEFS (peptide 1), ILPEDAPLD (peptide 2), DFDLPT (peptide 3), FDIDPG (peptide 4), and PLD (peptide 5) on the survival rate of MC3T3-E1 cells were investigated.

[0066] Figure 13 The effects of peptides DSVSEPQEFS (peptide 1), ILPEDAPLD (peptide 2), DFDLPT (peptide 3), FDIDPG (peptide 4) and PLD (peptide 5) on ALP activity in MC3T3-E1 cells were investigated.

[0067] Figure 14 The effect of the peptide FDIDPG on the expression of osteoblast differentiation-related genes in MC3T3-E1 cells; where A is the relative expression level of COL1A1 and B is the relative expression level of SPP1.

[0068] Figure 15 The effect of peptide FDIDPG on mineralization of MC3T3-E1 cells;

[0069] Figure 16The effect of the peptide FDIDPG on the expression of Runx2, Smad1, BMP-2, β1 integrin, p-FAK and p-ERK1 / 2 proteins in MC3T3-E1 cells. Detailed Implementation

[0070] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0071] Raw materials used in the examples:

[0072] Supercritical CO2 defatted egg yolk powder was purchased from Yibin Wild Resources Plant Chemical Co., Ltd. (Yibin, China).

[0073] Alkaline protease, catalog number FDG-2202, enzyme activity 200,000 U / g, purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. (Cangzhou, China).

[0074] The complex protease (Protamex) was purchased from Novozymes (Denmark), with an enzyme activity of 200,000 U / g;

[0075] The peptides used in the examples were synthesized by Nanjing Jietai Biotechnology Co., Ltd., and have a purity greater than 98%.

[0076] Test method:

[0077] 1. Cell Culture Methods

[0078] Cell resuscitation: MC3T3-E1 cells of passage 3-5 in good growth condition were used for the experiment. The culture medium was preheated to 37°C, with 15 mL of medium prepared in advance. Cryopreserved tubes containing 1 mL of cell suspension were rapidly thawed by shaking in a 37°C water bath. The cell suspension was then added to the culture dish via pipette, and the cells were mixed by shaking in a star-shaped motion. The culture dishes were placed in a 37°C, 5% CO2 incubator for 10 h.

[0079] Cell culture medium change: Change the medium every 2 days. First, observe the cell condition under a microscope. Then, use a 5 mL pipette to remove the original culture medium, wash with 5 mL of HBSS, add 10 mL of fresh culture medium, and place in an incubator.

[0080] Cell passage: When the cell density reaches 80%–90%, passage can be performed. The passage ratio is 1:(2–3). During passage, discard the old culture medium, rinse the cells twice with 5 mL of HBSS preheated to 37 °C, then add 1 mL of cell digestion solution to wet the surface. Digest in an incubator for 3 min, observing the digestion under a microscope. If most cells become rounded and detach, quickly return the cells to the operating table, add 3 mL of complete culture medium to stop digestion, and gently blow off the adherent cells. Aliquot 4 mL of the liquid into 2 mL of each 15 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, and resuspend the cells in 1 mL of culture medium. Then aliquot the cell suspension into culture dishes, mix well, and continue culturing in an incubator.

[0081] 2. Cell viability assay (MTT method)

[0082] Cell viability was determined using the MTT assay. MC3T3-E1 cells in logarithmic growth phase were harvested and cultured at a rate of 5 × 10⁻⁶ cells / year. 3 The cells were seeded into 96-well plates and incubated for 24 h. Afterward, the old culture medium was discarded, and 200 μL of culture medium containing different samples was added to each well. The plates were then incubated for another 24 h. Next, 20 μL of 5 mg / mL MTT solution was added to each well. After 4 h, the MTT-containing culture medium was discarded, and 150 μL of DMSO was added to each well. Finally, the absorbance at 490 nm was measured using a microplate reader. The cell viability of the control group was set as 100%. The cell viability of other experimental groups was calculated using the following formula:

[0083] Cell viability (%) = (OD) 实验组 / OD 对照组 ) × 100%.

[0084] 3. ALP activity detection

[0085] MC3T3-E1 cells in the logarithmic growth phase were harvested at a concentration of 1.5 × 10⁻⁶. 5 The cells were seeded into 6-well plates and incubated for 24 hours. The old culture medium was then discarded, and 2 mL of culture medium containing different samples was added to each well. The plates were then incubated for another 5 days. The culture medium was discarded, and ALP extraction buffer was added. Cell lysates were prepared using ultrasonic disruption. ALP activity was then measured according to the alkaline phosphatase kit instructions, and the results were calibrated using a BCA kit to determine protein content. Finally, the ALP activity of the control group was set to 100%, and the relative ALP activity of each experimental group was calculated using the following formula:

[0086] ALP activity (%) = (ALP activity in experimental group / ALP activity in control group) × 100%.

[0087] 4. Cell mineralization detection

[0088] MC3T3-E1 cells were grown at a rate of 1.5 × 10⁻⁶. 5 The cells were seeded into 12-well plates and incubated for 24 h. The old culture medium was then discarded, and 1 mL of differentiation medium (10 mM β-glycerophosphate sodium, 50 μg / mL ascorbic acid) containing different samples was added to each well. The cells were then incubated for another 21 days. MC3T3-E1 cells were treated with test samples at concentrations of 25, 50, and 100 μg / mL, respectively. The culture medium was then discarded, and the cells were washed once with HBSS, fixed with fixative for 20 min, and then washed three more times with HBSS. Finally, 1 mL of Alizarin Red S staining solution was added to each well, and the cells were stained at room temperature for 30 min. After thorough washing with distilled water, the cells were photographed and observed.

[0089] 5. Determination of molecular weight distribution

[0090] The sample was dissolved in 45% acetonitrile solution containing 0.1% TFA to form a 2 mg / mL sample solution. After filtration through a 0.22 μm aqueous filter, 10 μL was manually injected into a TSK gel G2000 SWXL column. Elution was performed with 45% acetonitrile (containing 0.1% TFA) at a flow rate of 0.5 mL / min. The detection wavelength was 220 nm. A standard curve was determined using elution times for molecular weights of 1173.3, 503.4, 365.8, and 118.2.

[0091] 6. Determination of amino acid composition

[0092] The determination of phenyl isothiocyanate (PITC) was performed using HPLC-PITC pre-column derivatization. 6 mg of sample was accurately weighed into a protein hydrolysis tube, 6 mL of 6.0 M HCl was added, and the tube was placed in a 110 ℃ oven for 24 h for hydrolysis. After hydrolysis, 1 mL of the hydrolysate was dried in a 42 ℃ oven for 4 h until the hydrochloric acid was fully evaporated. The solution was then reconstituted with distilled water and ready for analysis. 200 μL of the reconstituted sample was taken, and 100 μL of 0.1 M phenyl isothiocyanate and 100 μL of 1 M triethylamine were added. The mixture was thoroughly mixed and allowed to stand in the dark for 1 h. Then, 400 μL of n-hexane was added, vortexed for 1 min, and allowed to stand for 10 min. The supernatant was collected, filtered through a 0.22 μm organic filter membrane, and 10 μL was manually injected into an Agilent AdvanceBio AAA column (4.6 × 100 mm) for HPLC analysis.

[0093] Example 1: Preparation of egg yolk protein hydrolysate

[0094] 1. A method for preparing egg yolk protein hydrolysate, comprising the following steps:

[0095] (1) 5 g of defatted egg yolk powder (i.e., supercritical CO2 defatted egg yolk powder, hereinafter referred to as defatted egg yolk powder) was dissolved in 50 mL of deionized water, shaken for 10 min to mix, and ultrasonically pretreated at 300 W for 20 min to obtain a turbid liquid; the pH of the turbid liquid was adjusted to 11, and alkaline protease (Xiasheng) with E / S=1% (i.e., the mass ratio of alkaline protease to defatted egg yolk powder) was added for 1.5 h to enzymatically hydrolyze the liquid to obtain a first hydrolysate;

[0096] (2) Adjust the pH of the first enzymatic hydrolysate to 7, add a complex protease (Novozymes) with E / S=1% and continue enzymatic hydrolysis for 4 h to obtain the second enzymatic hydrolysate; adjust the pH of the second enzymatic hydrolysate to 7, boil in a water bath for 10 min to inactivate the enzyme, centrifuge at 5000 rpm for 10 min to collect the supernatant, freeze dry to obtain egg yolk protease hydrolysate (i.e., EYP), and store at -80℃.

[0097] 2. Effect of enzymatic hydrolysis time on yield

[0098] Based on step 1, the effect of enzymatic hydrolysis time (0-8 h) on the yield of the hydrolysate in step (2) was investigated, and the results are as follows: Figure 1 As shown.

[0099] The results showed that after 1.5 h of alkaline protease hydrolysis (i.e., 0 h), the yield of the hydrolysate was 34.64%. After adding the compound protease, the hydrolysis yield gradually increased with the extension of hydrolysis time. When hydrolyzing for 4 h to 8 h, the yield of the hydrolysate was higher than 50%.

[0100] 3. Detection of osteogenic function of egg yolk protein hydrolysate (EYP)

[0101] MTT assays showed that EYP at concentrations of 25–200 μg / mL did not inhibit the proliferation of osteoblast MC3T3-E1 cells. Therefore, the osteogenic function of EYP within this concentration range was assessed as follows:

[0102] (1) Effects of EYP concentration and induction time on osteoblast ALP activity

[0103] The effects of EYP concentrations of 20, 50, 100, and 200 μg / mL on ALP activity in MC3T3-E1 cells induced for 3, 4, and 5 days were examined, and the results are as follows: Figure 2 As shown.

[0104] The results showed that ALP activity (U / mg prot) increased significantly with increasing induction days. At the same induction days, the ALP activity of different concentrations of enzymatic hydrolysate varied significantly. 50 μg / mL showed the best effect at induction days 3 and 5, while 25 μg / mL showed the best effect at induction day 4. Compared to the control group, 50 μg / mL EYP increased ALP activity by 129.30%, 120.91%, and 50.56% at induction days 3, 4, and 5, respectively, demonstrating that 50 μg / mL EYP had the best effect on promoting ALP activity.

[0105] (2) Effect of EYP concentration on the expression of osteoblast differentiation-related proteins

[0106] MC3T3-E1 cells in logarithmic growth phase were cultured at a rate of 1.5 × 10⁻⁶. 5 The cells were seeded into 6-well plates, and after 24 h of culture, the old culture medium was discarded. 2 mL of EYP medium containing 20, 50, 100, and 200 μg / mL was added to each well, and the cells were incubated for another 3 days. Western blot was used to determine the expression level of Runx2 protein in osteoblasts, and the results are shown below. Figure 3 As shown.

[0107] Runx2 is an osteoblast-specific transcription factor that can regulate osteoblast differentiation, promote extracellular matrix formation, and thus accelerate bone formation.

[0108] The results showed that the relative expression level of Runx2 protein was increased at concentrations of 25, 50, and 100 μg / mL, indicating that EYP promoted osteogenic differentiation and bone formation at these concentrations. Among them, the optimal EYP treatment concentrations for Runx2 protein expression were 25 and 50 μg / mL.

[0109] (3) Effect of EYP concentration on the expression of osteoblast differentiation-related genes

[0110] MC3T3-E1 cells in logarithmic growth phase were cultured at a rate of 1.5 × 10⁻⁶. 5 The cells were seeded into 6-well plates, and after 24 h of culture, the old medium was discarded. 2 mL of EYP medium containing 20, 50, 100, and 200 μg / mL was added to each well, and the cells were incubated for another 5 days. The gene expression levels of COL1A1 and SPP1 in osteoblasts were measured using qRT-PCR, and the results are shown below. Figure 4 As shown.

[0111] The COL1A1 and SPP1 genes are responsible for guiding the synthesis of type I collagen and osteopontin, respectively. The efficacy of egg yolk protein hydrolysate in promoting osteogenic differentiation and bone organic matter synthesis can be evaluated by assessing the regulatory effect of EYP on the expression of COL1A1 and SPP1 genes.

[0112] The results showed that EYP significantly increased the expression of COL1A1 and SPP1 genes at concentrations of 50 and 100 μg / mL, while inhibiting SPP1 expression at concentrations of 200 and 400 μg / mL. Combined results from Western blot and qRT-PCR indicated that the optimal osteogenic concentration was 50–100 μg / mL.

[0113] (4) Effect of EYP concentration on osteoblast mineralization

[0114] The effects of EYP at concentrations of 25, 50, and 100 μg / mL on mineralization of MC3T3-E1 cells were examined, and the results are as follows: Figure 5 As shown.

[0115] The results showed that a small number of uneven calcium nodules appeared in the control group. The number of nodules increased at a concentration of 50 μg / mL, and the mineralized area was largest and most uniformly distributed at this concentration. At a concentration of 100 μg / mL, the nodules were uniformly distributed but the color depth decreased. In other words, EYP can promote the mineralization of MC3T3-E1 cells at different doses, exhibiting a dose-response relationship, with 50 μg / mL being the optimal dose.

[0116] 4. Comparison with existing reported enzymatic hydrolysis products

[0117] The enzymatic hydrolysate prepared in step 1 was compared with the egg yolk enzymatic hydrolysate reported in existing patents, and the results are shown in Table 1.

[0118] Table 1. Comparison of the properties of different enzymatic hydrolysis products

[0119]

[0120] The egg yolk hydrolysate prepared by the two-step enzymatic hydrolysis method in Example 1 showed significantly higher osteogenic differentiation-promoting activity (ALP activity, a biomarker) than the hydrolysate prepared by single-enzyme hydrolysis (previous technologies 2, 3, and 4). However, the effects of the hydrolysate prepared by combined enzymatic hydrolysis (previous technologies 5 and 6) could not be compared because the ALP activity was not provided. It can be inferred that the molecular weight of the hydrolysate prepared by Example 1 was greater than that of prior technologies 5 and 6.

[0121] Both Example 1 and Prior Art 1 employ a two-step enzymatic hydrolysis method. However, Prior Art 1 only describes the method of first hydrolyzing with alcalase and then adding flavor protease, while Example 1 employs a two-step enzymatic hydrolysis method of first hydrolyzing with alkaline protease and then adding complex protease. The proteases used in the two methods are very different.

[0122] The amino acid composition of the enzymatic hydrolysates prepared in Example 1 and the prior art 1 is shown in Table 2. It can be seen that the amino acid composition of the enzymatic hydrolysates of the two is very different.

[0123] Table 2. Amino acid composition of enzymatic hydrolysates from Example 1 and prior art 1

[0124]

[0125] Meanwhile, the enzymatic hydrolysate of Example 1 showed osteogenic differentiation-promoting activity (ALP enzyme activity at a comparative concentration of 50 μg / mL) that was higher than or equivalent to that of Comparative Example 1 (the osteoblast induction differentiation period in Example 1 was 5 days, while that in Comparative Example 1 was 8 days; as the osteoblast induction differentiation period increased, the ALP enzyme activity increased), and the yield of the enzymatic hydrolysate was much higher than that of the prior art 1.

[0126] Example 2: Preparation of egg yolk protein peptide composition

[0127] The egg yolk protein hydrolysate prepared in Example 1 was further separated and purified using macroporous resin XA-1600, as follows:

[0128] Wet macroporous resin XA-1600 was packed into a chromatography column (2.5 cm × 40 cm), and the column was rinsed with distilled water until no change was detected at 220 nm. 1 g of enzymatic hydrolysate was weighed and prepared into a 100 mg / mL solution for manual loading.

[0129] The peristaltic pump flow rate was set to 3 mL / min. The chromatography column was dynamically desorbed sequentially with deionized water, 20% (v / v) ethanol aqueous solution, 40% ethanol aqueous solution, and 80% ethanol aqueous solution, for 20 min with each eluent. Each eluent fraction was collected, and the fractions were rotary evaporated under vacuum at 55°C until no ethanol remained. The fractions obtained from the desorption set were identified as egg yolk peptides and named EYP1, EYP2, EYP3, and EYP4, respectively.

[0130] Following the method described in Example 1, the osteogenic function of EYP1, EYP2, EYP3, and EYP4 at a concentration of 50 μg / mL was detected, and the results are as follows: Figure 6 As shown in the figure. The results indicate that EYP1, EYP2, EYP3, and EYP4 all have a certain ability to promote bone differentiation, with EYP2 showing the best effect. Since peptide separation is carried out in descending order of polarity, EYP2 belongs to the weakly polar component.

[0131] Example 3: Preparation of egg yolk protein peptide composition

[0132] The egg yolk protein peptide composition EYP2 prepared in Example 2 was further separated and purified using Sephadex G-15 dextran gel, as follows:

[0133] EYP2 was prepared into a 100 mg / mL solution, filtered through a 0.45 μm aqueous filter membrane, and 2 mL was manually loaded onto a Sephadex G-15 column (2.5 cm × 40 cm) for separation.

[0134] The eluent was deionized water, the flow rate was controlled by a peristaltic pump at 1.0 mL / min, the elution time was 180 min, and the elution volume was 180 mL. The absorbance was monitored at 220 nm, the spectrum was recorded, and the sample components were collected and named EYP2-1, EYP2-2, and EYP2-3, respectively.

[0135] Following the method described in Example 1, the osteogenic function of EYP2-1, EYP2-2, and EYP2-3 at a concentration of 50 μg / mL was assessed, and the results are as follows: Figure 7 As shown in the figure. The results indicate that EYP2-1 has the best ability to promote bone differentiation.

[0136] Example 4: Preparation of egg yolk protein peptide composition

[0137] The egg yolk protein peptide composition EYP2-1 prepared in Example 3 was further separated and purified using a dextran gel SP Spheadex C-25, as follows:

[0138] Prepare a 0.02 M, pH 4 sodium acetate buffer solution to fully swell the dextran gel. After washing away the suspended particles, pack the column (2.5 cm × 40 cm). Rinse the column with the buffer solution until it reaches equilibrium. Once the gel bed inside the column is stable, ion exchange chromatography can be performed.

[0139] EYP2-1 was prepared into a 100 mg / mL solution, filtered through a 0.45 μm aqueous filter membrane, and 0.7 mL was manually loaded. A gradient elution was performed using acetate buffer, 0.05, 0.1, 0.2, and 0.4 M NaCl / acetic buffer as eluents, eluting for 120 min with each eluent at a flow rate of 1.5 mL / min for a total elution volume of 180 mL. The absorbance was monitored at 220 nm, yielding two absorption peaks (obtained from acetate buffer and 0.2 M NaCl / acetic buffer elution, respectively). These two eluted fractions were collected and concentrated separately.

[0140] The two elution fractions were then desalted using the following method: the Sephadex C-25 elution fraction was concentrated to 2 mL and injected into a YMC ODS-A C18 column (1.0 cm × 10 cm); water and 50% methanol aqueous solution were used for elution for 20 min each, at a flow rate of 1 mL / min; salts were completely eluted in the first 10 min, and the sample fraction was collected after 10 min. The two desalted elution fractions were named EYP2-1-1 and EYP2-1-2, respectively.

[0141] Following the method described in Example 1, the osteogenic function of EYP2-1-1 and EYP2-1-2 at a concentration of 50 μg / mL was assessed, and the results are as follows: Figure 8 As shown.

[0142] The results showed that the expression level of Runx2 protein in EYP2-1-1 was significantly higher than that in EYP2-1, indicating that the active peptide sequence was rich in acidic amino acids.

[0143] Example 5: Detection of molecular weight distribution of egg yolk protein peptide composition

[0144] The egg yolk protein peptide compositions EYP, EYP2, EYP2-1, and EYP2-1-1 prepared in Examples 2-4 were analyzed for their molecular weight distribution. The results are as follows: Figure 9 As shown in Table 3.

[0145] Table 3 Molecular weight distribution of EYP, EYP2, EYP2-1, and EYP2-1-1

[0146]

[0147] The results showed that peptides <500 Da were generally enriched. EYP2-1 contained a relatively large number of macromolecular components, which is consistent with the fact that it was the first component to elute in Sephadex G15. The enrichment trend indicates that most of the highly active components originated from oligopeptides <500 Da.

[0148] Example 6: Identification of egg yolk protein peptides

[0149] 1. Identification and synthesis of polypeptide sequences

[0150] The effective peptide components of EYP2-1-1 prepared in Example 4 were identified by LC-MS / MS. Using Uniprot chicken egg yolk protein as a database, the mass spectrometry identification was compared with the database to screen 5 peptides with high peptide abundance and amino acid composition characteristics that conform to the characteristics of Examples 2, 3 and 4. The results are shown in Table 4.

[0151] The total ion chromatogram of the EYP2-1-1 fraction and the primary mass spectrum containing the above five peptides (retention times of 22.72–28.30 min and 33.22–38.79 min) are shown below. Figure 10 As shown; the secondary mass spectra used to identify these peptide sequences are as follows. Figure 11As shown, the peptides include (A) DSVSEPQEFS (peptide-1), (B) ILPEDAPLD (peptide-2), (C) DFDLPT (peptide-3), (D) FDIDPG (peptide-4), and (E) PLD (peptide-5). These peptides were synthesized by a biotechnology company using a solid-phase synthesis method to assess their bioactivity and explore the molecular regulatory mechanisms of osteogenic peptides.

[0152] Table 4. Screening of peptide results

[0153]

[0154] 2. Peptide performance testing

[0155] The osteogenic properties of the polypeptides DSVSEPQEFS, ILPEDAPLD, DFDLPT, FDIDPG, and PLD prepared in Example 1 were tested using the same method as in Example 1, as follows:

[0156] (1) Effects of polypeptides on cell viability

[0157] The effect of 50, 100, and 200 μM peptides on the survival rate of MC3T3-E1 cells was detected by MTT assay, and the results are as follows: Figure 12 As shown in the figure. The results showed that DFDLPT (peptide 3) had a significant proliferative effect on MC3T3-E1 cells at concentrations of 50–200 μM, with a cell viability of 121.23% at a concentration of 100 μM. The other four peptides showed no significant difference from the control group after addition.

[0158] (2) Effect of peptides on ALP activity

[0159] The effects of 50, 100, and 200 μM peptides on ALP activity in MC3T3-E1 cells were measured, with the ALP activity of the control group considered as 100%. Results are as follows: Figure 13 As shown, the results indicate that FDIDPG has a good effect on promoting osteoblast differentiation. At the three induction concentrations of 50, 100, and 200 μM, the ALP levels in cells were significantly higher than those in the control group, and a good dose-response relationship was observed. At the 200 μM concentration, the relative ALP activity reached 136.15%. The other four peptides did not show significant activity, so only FDIDPG was tested for subsequent performance.

[0160] (3) Effects of FDIDPG on the expression of osteoblast differentiation-related genes

[0161] The effects of 50, 100, and 200 μM FDIDPG on the expression of osteoblast differentiation-related genes were examined, with the gene expression level of the control group representing 100%. Results are as follows: Figure 14As shown, the results indicate that FDIDPG at 50, 100, and 200 μM significantly upregulated the expression of COL1A1 and SPP1 genes; among them, the relative expression of COL1A1 gene was highest at 200 μM induction, reaching 219.71%, and that of SPP1 reached 295.44%.

[0162] (4) Effects of FDIDPG on osteoblast mineralization

[0163] The effects of 50, 100, and 200 μM FDIDPG on osteoblast mineralization were investigated, and the results are as follows: Figure 15 As shown in the figure, the results indicate that with increasing FDIDPG concentration, the mineralized area of ​​cells under the microscope gradually increases, and uniform and clear calcium nodule granules are gradually formed. The mineralization level reaches its highest point when the concentration reaches 200 μM.

[0164] (5) Effects of FDIDPG on the expression of key signaling proteins in osteoblasts

[0165] The effects of 100 and 200 μM FDIDPG on the expression levels of key signaling proteins (Runx2, Smad1, BMP-2, β1 integrin, p-FAK, and p-ERK) were examined, and the results are as follows: Figure 16 As shown in the figure. The results indicate that FDIDPG has a good activity in promoting the expression of the osteocalcin protein Runx2. When the concentration of FDIDPG is 100 μM, the relative expression of Runx2 protein is significantly increased to 198.60% compared with the control group.

[0166] Meanwhile, FDIDPG has the function of upregulating Smad1 protein, and its expression level can be increased to 159.58% at a concentration of 200 μM, which proves that FDIDPG may promote Runx2 protein expression through the BMP-Smads or TGF-β / Smads signaling pathway.

[0167] Furthermore, the relative expression of BMP-2 protein was significantly enhanced. This indicates that FDIDPG stimulates the BMP / Smads signaling pathway to regulate the expression of Runx2 protein by binding to the BMP-2 receptor, thereby producing a significant osteogenic effect. β1 integrin was also regulated by FDIDPG, which affected the expression of p-FAK and p-ERK, resulting in the highest levels of β1 integrin, p-FAK, and p-ERK reaching 129.01%, 128.27%, and 112.59%, respectively, all significantly higher than the original levels.

[0168] The results in summary indicate that FDIDPG can promote the expression of key signaling proteins in osteoblasts.

[0169] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An egg yolk protein peptide, characterized by, The egg yolk protein peptide is FDIDPG or DFDLPT, and the amino acid sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively.

2. A process for the preparation of egg yolk protease digests containing egg yolk protein peptides having the amino acid sequences shown in SEQ ID NO. 1 and / or SEQ ID NO. 2, respectively, characterized in that, The method comprises the steps of: (1) mixing defatted egg yolk powder with water, ultrasonic treatment to obtain a turbid solution; adjusting the pH of the turbid solution to 10-11, adding alkaline protease with an E / S ratio of 0.5-1.5%, and enzymolysis for 1.0-1.5 h to obtain a first enzymolysis solution; (2) adjusting the pH of the first enzymolysis solution to 6.5-7, adding complex protease with an E / S ratio of 0.8-1.5%, and enzymolysis for 2-8 h to obtain a second enzymolysis solution; adjusting the pH of the second enzymolysis solution to 6.5-7, inactivating the enzyme, centrifuging to collect the supernatant, and freeze-drying to obtain the egg yolk protein enzymolysis product; Wherein, the alkaline protease is purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., and the item number is FDG 2202; Complex protease is complex protease Protamex purchased from Novozymes.

3. The method of claim 2, wherein, In step (1), the ratio of the amount of defatted egg yolk powder to water is 1 g:7-15 mL.

4. The method of claim 2, wherein, In step (1), the ultrasonic treatment is 100-300 W for 10-20 min.

5. The egg yolk protein enzymolysis product prepared by the method of any one of claims 2-4.

6. A method of preparing an egg yolk protein peptide composition containing the egg yolk protein peptide according to claim 1, characterized by, The method comprises the steps of: (1) Defatted egg yolk powder is mixed with water, ultrasonic treatment to obtain a turbid solution; the pH of the turbid solution is adjusted to 10-11, and alkaline protease with an E / S of 0.5-1.5% is added, and enzymolysis is carried out for 1-1.5 h to obtain a first enzymolysis solution; wherein the alkaline protease is purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., with a product number FDG 2202; (2) adjusting the pH of the first enzymolysis solution to 6.5-7, adding complex protease with an E / S ratio of 0.8-1.5%, and enzymolysis for 2-8 h to obtain a second enzymolysis solution; adjusting the pH of the second enzymolysis solution to 6.5-7, inactivating the enzyme, centrifuging to collect the supernatant, and freeze-drying to obtain the egg yolk protein enzymolysis product; wherein the complex protease is Protamex purchased from Novozymes; (3) separating the egg yolk protein enzymolysis product by using macroporous resin XA-1600, eluting with an aqueous ethanol solution with a volume fraction of 0-80%, and collecting different gradient eluates for drying to obtain an egg yolk protein peptide composition obtained by first purification and separation.

7. The method of claim 6, wherein, In step (3), deionized water, an aqueous ethanol solution with a volume fraction of 20%, an aqueous ethanol solution with a volume fraction of 40%, and an aqueous ethanol solution with a volume fraction of 80% are used in sequence for elution, the elution flow rate is 3-5 mL / min, and one column volume is eluted at each gradient.

8. The method of claim 6, wherein, In step (3), the egg yolk protein peptide composition obtained by first purification and separation is eluted by using an aqueous ethanol solution with a volume fraction of 20% and dried.

9. The method of claim 6, wherein, The egg yolk protein peptide composition obtained by first purification and separation can also be further separated by using dextran gel Sephadex G-15, eluted with water, and monitored for absorbance at 220 nm to collect an egg yolk protein peptide composition obtained by second purification and separation.

10. The method of claim 9, wherein, The elution flow rate is 1-2 mL / min, the elution time is 90-180 min, and the elution volume is 180 mL; the eluate collected from the first absorption peak appearing on the elution curve is the egg yolk protein peptide composition obtained by second purification and separation; the elution time period of the first absorption peak appearing on the elution curve is within the first 60 min.

11. The method of claim 9, wherein, The egg yolk protein peptide composition obtained by second purification and separation can also be further separated by using dextran gel SP Spheadex C-25, gradient eluted with a 0-0.4 M NaCl / acetic acid buffer, monitored for absorbance at 220 nm, and collected to obtain an egg yolk protein peptide composition obtained by third purification and separation; The elution flow rate is 1-1.5 mL / min, and the elution time of each eluent is 120-180 min; the eluate collected in the time period of deionized water elution is the egg yolk protein peptide composition purified and separated for three times, and the time period of deionized water elution is 120 min.

12. The egg yolk protein peptide composition prepared by the method of any one of claims 6-11.

13. A pharmaceutical or nutraceutical product, characterized in that, The pharmaceutical product or health care product contains the egg yolk protein peptide of claim 1, the egg yolk protein enzymatic hydrolysate of claim 5, or the egg yolk protein peptide composition of claim 12.

14. The pharmaceutical or nutraceutical product according to claim 13, characterized in that, The pharmaceutical product further contains a pharmaceutically acceptable pharmaceutical excipient; the pharmaceutical excipient refers to a conventional drug carrier in the pharmaceutical field.

15. The pharmaceutical or nutraceutical product according to claim 13, characterized in that, The dosage form of the pharmaceutical product includes oral dosage form, injection dosage form, and inhalation dosage form.

16. The pharmaceutical or nutraceutical product according to claim 15, characterized in that, The oral dosage form includes tablets, capsules, granules, oral liquids, and oral suspensions.

17. The pharmaceutical or nutraceutical product according to claim 15, characterized in that, The injection dosage form includes injection liquids and injection powders.

18. The drug product or health product of claim 15, wherein, The inhalation dosage form includes aerosols and powder aerosols.

19. Use of the egg yolk protein peptide of claim 1, the egg yolk protein enzymatic hydrolysate of claim 5, or the egg yolk protein peptide composition of claim 12 in the preparation of a product for promoting bone growth or improving bone density.

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