A hydrolyzed egg yolk powder rich in polypeptide growth factor-like substances, its preparation method and applications

Abstract

The present invention discloses a hydrolyzed egg yolk powder rich in growth factor-like polypeptides, its preparation method and application. The present invention relates to the fields of biotechnology and nutritional health, specifically to a hydrolyzed egg yolk powder rich in growth factor-like polypeptides and its application as a medicament for promoting bone growth. The hydrolyzed egg yolk powder is obtained by extracting from egg yolk through a specific enzymatic hydrolysis technology and is rich in growth factor-like polypeptides with significant bone formation promoting functions. The hydrolyzed egg yolk powder and growth factor-like polypeptides in the present invention can be used as pharmaceutical ingredients to effectively promote bone growth and have significant effects on treating or preventing diseases related to bone health such as osteogenesis imperfecta, osteoporosis and osteoarthritis, etc.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a hydrolyzed egg yolk powder rich in growth factor-like polypeptides, a preparation method thereof and an application thereof, especially an application in promoting bone growth. Background Art

[0002] With the acceleration of the pace of life and the aggravation of the trend of population aging, bone health problems have been increasingly concerned. Bone diseases such as osteogenesis imperfecta, osteoporosis and osteoarthritis seriously affect the quality of life of patients. At present, although there are various treatment methods, the curative effects are not ideal, and there are problems such as large side effects and long treatment cycles. Therefore, developing new, safe and effective bone growth promoters has important clinical significance and market demand.

[0003] As a natural nutritional supplement, hydrolyzed egg yolk powder has attracted much attention because it is rich in various bioactive substances. Among them, growth factor-like polypeptides, as an important class of bioactive substances, have functions such as promoting cell proliferation, differentiation and tissue repair, and have a significant promoting effect on bone growth. However, there is currently no hydrolyzed egg yolk powder product rich in growth factor-like polypeptides on the market. Summary of the Invention

[0004] Object of the Invention: In order to overcome the deficiencies in the prior art, the present invention provides a hydrolyzed egg yolk powder rich in growth factor-like polypeptides, a preparation method thereof and an application thereof. The polypeptide of the present invention is derived from hydrolyzed egg yolk powder and is obtained by extracting from egg yolk powder through specific enzymatic hydrolysis or hydrolysis technology. This polypeptide is rich in the activity of human epidermal growth factor-like, can simulate the action of human epidermal growth factor (EGF), stimulate the proliferation and differentiation of osteoblasts, promote the synthesis and secretion of bone matrix, thereby accelerating bone growth and repair. At the same time, this polypeptide can also inhibit the activity of osteoclasts, reduce bone loss, and improve bone density and bone strength.

[0005] Technical Solution: To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The first object of the present invention is to provide a growth factor-like polypeptide, and the growth factor-like polypeptide includes at least one of the following:

[0007] Polypeptide I, the amino acid sequence is FRTPPFGGF,

[0008] Polypeptide II, the amino acid sequence is HGFWPRDPFTPS,

[0009] Polypeptide III, the amino acid sequence is PFAEYPTYK,

[0010] Polypeptide IV, the amino acid sequence is SRPILPIYLK,

[0011] Polypeptide V, with the amino acid sequence of WPRDPFTR.

[0012] The second object of the present invention is to provide a hydrolyzed egg yolk powder rich in polypeptide-like growth factors. The hydrolyzed egg yolk powder contains polypeptide-like growth factors, and the polypeptide-like growth factors include:

[0013] Polypeptide I, with the amino acid sequence of FRTPPFGGF,

[0014] Polypeptide II, with the amino acid sequence of HGFWPRDPFTPS,

[0015] Polypeptide III, with the amino acid sequence of PFAEYPTYK,

[0016] Polypeptide IV, with the amino acid sequence of SRPILPIYLK,

[0017] Polypeptide V, with the amino acid sequence of WPRDPFTR.

[0018] Optionally, in some embodiments of the present invention, the molecular weight of all peptide segments of the hydrolyzed egg yolk powder is less than 250000 Da.

[0019] Further optionally, in some embodiments of the present invention, the molecular weight distribution of more than 97% of the peptide segments of the hydrolyzed egg yolk powder is concentrated in the range of 180 - 2000 Da,

[0020] Even more optionally, in some embodiments of the present invention, the molecular weight distribution of 97% - 98% of the peptide segments of the hydrolyzed egg yolk powder is concentrated in the range of 180 - 2000 Da.

[0021] The third object of the present invention is to provide a method for preparing a hydrolyzed egg yolk powder rich in polypeptide-like growth factors, which is characterized by including the following steps:

[0022] (1) Pretreatment: After dissolving the defatted egg yolk powder, it is subjected to alkali heat treatment at 50°C - 90°C under the condition of pH 9 - 10, and then wet pulverization treatment is carried out to obtain a pretreated egg yolk powder solution;

[0023] (2) The pretreated egg yolk powder solution obtained in step (1) is adjusted to pH 9 - 10, and then alkaline protease is added for enzymatic hydrolysis at 55°C ± 5°C to obtain an enzymatic hydrolysate;

[0024] (3) The enzymatic hydrolysate obtained in step (2) is centrifuged to obtain a supernatant;

[0025] (4) The supernatant obtained in step (3) is subjected to nanofiltration for desalination and concentration to obtain a concentrated solution;

[0026] (5) The concentrated solution obtained in step (4) is dried to obtain a hydrolyzed egg yolk powder.

[0027] Optionally, in some embodiments of the present invention, in step (1), the protein content of the defatted egg yolk powder is 60%-90%.

[0028] Optionally, in some embodiments of the present invention, in step (1), the defatted egg yolk powder may be a commercially available defatted egg yolk powder or a low-value defatted egg yolk powder after industrial extraction of lecithin, and the protein content is 60%-90%.

[0029] Optionally, in some embodiments of the present invention, in step (1), the dissolution concentration of the defatted egg yolk powder is 6-10 ml of water / g of defatted egg yolk powder. Further preferably, it is 6-8 ml of water / g of defatted egg yolk powder.

[0030] Optionally, in some embodiments of the present invention, the time of alkali heat treatment is 15-30 min.

[0031] Further optionally, in some embodiments of the present invention, the time of alkali heat treatment is 15-20 min. Further preferably, it is 15 min.

[0032] Optionally, in some embodiments of the present invention, the pH of the alkali heat treatment is 9-9.5. Further preferably, it is 9.5.

[0033] Optionally, in some embodiments of the present invention, the temperature of the alkali heat treatment is 60°C-90°C.

[0034] Further optionally, in some embodiments of the present invention, the temperature of the alkali heat treatment is 70°C-90°C.

[0035] Even more optionally, in some embodiments of the present invention, the temperature of the alkali heat treatment is 80°C-90°C. Further preferably, it is 90°C.

[0036] Optionally, in some embodiments of the present invention, in step (1), the wet grinding treatment includes wet grinding with a colloid mill to obtain a defatted egg yolk powder homogenate.

[0037] Optionally, in some embodiments of the present invention, in step (2), the alkaline protease includes Alcalase enzyme and / or trypsin, etc.

[0038] Optionally, in some embodiments of the present invention, the time of enzymatic hydrolysis is 2-6 h.

[0039] Further optionally, in some embodiments of the present invention, the time of enzymatic hydrolysis is 3-5 h. Further preferably, it is 4 h.

[0040] Optionally, in some embodiments of the present invention, the addition amount of the alkaline protease is 1-3 wt% of defatted egg yolk powder.

[0041] Further optionally, in some embodiments of the present invention, the addition amount of the alkaline protease is 1.5-2.5 wt% of defatted egg yolk powder. More preferably, it is 2.0 wt% of defatted egg yolk powder.

[0042] Optionally, in some embodiments of the present invention, in step (3), the centrifugation includes centrifugation using a tubular centrifuge.

[0043] Further optionally, in some embodiments of the present invention, the centrifugation rate is 3000-8000 rpm, and the centrifugation time is 15-35 min.

[0044] Even more optionally, in some embodiments of the present invention, the centrifugation rate is 5000 rpm, and the centrifugation time is 25 min.

[0045] Optionally, in some embodiments of the present invention, in step (4), the nanofiltration desalination and concentration include subjecting the supernatant to nanofiltration desalination treatment through a 150 Da membrane.

[0046] Optionally, in some embodiments of the present invention, in step (5), the drying includes spray drying or freeze drying.

[0047] Further optionally, in some embodiments of the present invention, the spray drying includes an inlet air temperature of 180 °C and an outlet air temperature of 80 °C.

[0048] Optionally, in some embodiments of the present invention, the hydrolyzed egg yolk powder rich in growth factor-like polypeptides in the present invention can be prepared by the following steps:

[0049] (1) Dissolve defatted egg yolk powder in water to 6-8 ml of water per g of defatted egg yolk powder, add sodium hydroxide to adjust the pH to 9.5, perform heat treatment at 90 °C for 15-30 min, and then perform wet grinding treatment to obtain a defatted egg yolk powder homogenate;

[0050] (2) After adjusting the obtained defatted egg yolk powder homogenate to pH 9-10, add an alkaline protease for enzymatic hydrolysis;

[0051] (3) After enzymatic hydrolysis, centrifuge to obtain a supernatant;

[0052] (4) Desalt the supernatant obtained in step (3);

[0053] (5) Spray dry the final supernatant to obtain hydrolyzed egg yolk powder.

[0054] (6) Using separation and purification techniques such as chromatography and electrophoresis, the target polypeptide of human epidermal growth factor-like is separated from the crude polypeptide product.

[0055] The fourth object of the present invention is to provide the application of the hydrolyzed egg yolk powder described above, or the hydrolyzed egg yolk powder prepared by any of the above methods, in the preparation of drugs with the function of promoting bone growth.

[0056] The fifth object of the present invention is to provide a polypeptide of human epidermal growth factor-like, and the amino acid sequence of the polypeptide is as follows:

[0057] Polypeptide I, the amino acid sequence is FRTPPFGGF,

[0058] Polypeptide II, the amino acid sequence is HGFWPRDPFTPS,

[0059] Polypeptide III, the amino acid sequence is PFAEYPTYK,

[0060] Polypeptide IV, the amino acid sequence is SRPILPIYLK,

[0061] Polypeptide V, the amino acid sequence is WPRDPFTR.

[0062] Optionally, in some embodiments of the present invention, the polypeptide is obtained by purification from the hydrolyzed egg yolk powder, or the polypeptide is synthesized separately.

[0063] The fifth object of the present invention is to provide the application of the polypeptide of human epidermal growth factor-like described above in the preparation of drugs with the function of promoting bone growth.

[0064] Beneficial effects: The hydrolyzed egg yolk powder of the present invention has the following advantages:

[0065] (1) Rich in growth factor-like polypeptides, with a significant bone formation promoting function;

[0066] (2) Natural source, safe and without side effects;

[0067] (3) Can be used as a drug ingredient, convenient and flexible;

[0068] (4) Has a significant effect on the treatment or prevention of diseases related to bone health.

[0069] (5) The polypeptide of the present invention has a significant effect on promoting bone health. It can be used as an active ingredient of drugs for the prevention and treatment of bone diseases such as osteoporosis and fractures, and to promote bone health and growth and development. Description of the Drawings

[0070] Figure 1 It is a map of TIC & BPC of the nano-HPLC-MS / MS detection result.

[0071] Figure 2 It is the molecular weight map of polypeptides in the hydrolyzed egg yolk powder product in Example 1.

[0072] Figure 3 It is the molecular weight map of polypeptides in the hydrolyzed egg yolk powder product in Example 2.

[0073] Figure 4 It is the specific implementation manner of the effects of polypeptides I-V on the cell proliferation rate of MC3T3-E1.

[0074] Figure 5 It is the proliferation promotion effect of polypeptides I-V and hydrolyzed egg yolk powder on osteoprogenitor cells. Specific implementation manner

[0075] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the specific material ratios, process conditions and their results described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0076] On the one hand, the present invention provides a human epidermal growth factor analog (growth factor-like polypeptide), which is rich in the activity of human epidermal growth factor-like, can simulate the action of human epidermal growth factor (EGF), and can promote cell proliferation and differentiation. Specifically, the amino acid sequence of the polypeptide is at least one of the following:

[0077] Polypeptide I, with the amino acid sequence of FRTPPFGGF,

[0078] Polypeptide II, with the amino acid sequence of HGFWPRDPFTPS,

[0079] Polypeptide III, with the amino acid sequence of PFAEYPTYK,

[0080] Polypeptide IV, with the amino acid sequence of SRPILPIYLK,

[0081] Polypeptide V, with the amino acid sequence of WPRDPFTR.

[0082] On the other hand, the present invention provides a hydrolyzed egg yolk powder rich in human epidermal growth factor analogs (growth factor-like polypeptides), which are rich in the activity of human epidermal growth factor-like, can simulate the action of human epidermal growth factor (EGF), and can promote cell proliferation and differentiation. Specifically, the amino acid sequence of the polypeptide is as follows:

[0083] Polypeptide I, with the amino acid sequence of FRTPPFGGF,

[0084] Polypeptide II, with the amino acid sequence HGFWPRDPFTPS,

[0085] Polypeptide III, with the amino acid sequence PFAEYPTYK,

[0086] Polypeptide IV, with the amino acid sequence SRPILPIYLK,

[0087] Polypeptide V, with the amino acid sequence WPRDPFTR.

[0088] Another aspect of the present invention provides a method for preparing a hydrolyzed egg yolk powder rich in human epidermal growth factor analogs (growth factor-like polypeptides), comprising the following steps:

[0089] (1) Pretreatment: Dissolve defatted egg yolk powder in water, add sodium hydroxide to adjust the pH to 9 - 10, perform heat treatment at 50°C - 90°C for 15 - 30 minutes, and then perform wet grinding treatment to obtain a defatted egg yolk powder homogenate (also referred to as a pretreated egg yolk powder solution);

[0090] (2) Adjust the pH of the obtained defatted egg yolk powder homogenate to 9 - 10, and then add alkaline protease for enzymatic hydrolysis;

[0091] (3) After enzymatic hydrolysis, centrifuge to obtain the supernatant;

[0092] (4) Subject the supernatant obtained in step (3) to nanofiltration desalination through a 150 Da membrane;

[0093] (5) Spray-dry the final supernatant to obtain the product hydrolyzed egg yolk powder.

[0094] In one embodiment of the present invention, the defatted egg yolk powder can be commercially available defatted egg yolk powder or low-value defatted egg yolk powder after industrial extraction of lecithin, with a protein content of 60% - 90%.

[0095] Another object of the present invention is to provide a polypeptide similar to human epidermal growth factor, which can be purified from hydrolyzed egg yolk powder or synthesized separately.

[0096] The polypeptide similar to human epidermal growth factor obtained in the present invention has the function of human epidermal growth factor, can promote cell proliferation and differentiation, including but not limited to osteoblasts, and the polypeptide can stimulate the proliferation and differentiation of osteoblasts, promote the synthesis and secretion of bone matrix, thereby accelerating bone growth and repair.

[0097] The hydrolyzed egg yolk powder and polypeptide obtained in the present invention can promote bone formation and can be applied to the preparation of drugs with the function of promoting bone growth.

[0098] In the embodiments of the present invention, the structural identification of polypeptides is all carried out by nano-HPLC-MS / MS analysis. The specific analysis method is as follows: For each sample, about 200 ng of total peptides are separated by the nano-UPLC liquid phase system Evosepone and then combined with a mass spectrometer (timsTOFPro2) equipped with a nano-ion source for data acquisition. Chromatographic separation is carried out using a 150 μm ID × 15 cm reversed-phase chromatographic column (PePSep C18, 1.9 μm, 150 μm × 15 cm, Bruker, Germany). The mobile phase uses an acetonitrile-water-formic acid system, where mobile phase A is a 0.1% formic acid aqueous solution and phase B is a 0.1% formic acid acetonitrile solution. After the chromatographic column is equilibrated with 100% of phase A, the sample is directly injected onto the chromatographic column by an autosampler and then gradient separated by the chromatographic column with a gradient duration of 44 min.

[0099] In the embodiments of the present invention, the mass spectrometer uses the DDAPaSEF mode for DDA data acquisition, and the scanning range is from 100 - 1700 m / z. During the PASEF MS / MS scan, the collision energy linearly increases with the ion mobility, rising from 20 eV (1 / K0 = 0.6 Vs / cm2) to 59 eV (1 / K0 = 1.6 Vs / cm2). The raw data files are searched against a database using the Pulsar search engine of the SpectroMine (4.2.230428.52329; Biognosys AG) software, and qualitative analysis is carried out after the search. The TIC & BPC results of nano-HPLC-MS / MS are as Figure 1 shown

[0100] In the embodiments of the present invention, the egg yolk polypeptides are screened for peptide segments that may have osteogenic activity by computer virtual screening. The specific process is as follows:

[0101] Molecular docking is performed on all the peptide segments identified by mass spectrometry: Human epidermal growth factor receptor (EGFR) is selected as the receptor protein, and human epidermal growth factor (EGF) is used as the original ligand. Homology modeling is used to find the active center of EGFR, and AutoDock Vina is used to perform molecular docking on EGFR and egg yolk protein peptides. They are arranged in ascending order according to the binding energy, and the top five polypeptides (binding energy < -7) are selected. According to the literature, a binding energy of AutoDock Vina less than -7 is considered reliable.

[0102] In the embodiments of the present invention, the method for verifying the osteoblast proliferation activity of polypeptides is as follows:

[0103] Mouse pre-osteoblast cell line MC3T3-E1 cells were inoculated into 96-well plates at a density of 2×104 cells / mL, 100 μL per well. After culturing at a constant temperature for 24 h, the original medium was removed. Subsequently, a series of polypeptide solutions with gradient mass concentrations were prepared and added to the 96-well plates at 200 μL per well. The cells were continuously cultured in an incubator at 37 °C with a CO2 volume fraction of 5% for 48 h. The blank group was added with an equal volume of medium. The proliferation rate was measured by the CCK-8 method, and each experiment was repeated 4 times in parallel. The relative proliferation rate of pre-osteoblasts was calculated according to the following formula.

[0104]

[0105] In the formula: A sample is the absorbance of the sample solution; A blank is the absorbance of the blank group solution.

[0106] In the embodiments of the present invention, the peptide in the present invention is a peptide composed of the amino acid sequences represented by FRTPPFGGF (peptide I, as shown in SEQ ID 01), HGFWPRDPFTPS (peptide II, as shown in SEQ ID 02), PFAEYPTYK (peptide III, as shown in SEQ ID 03), SRPILPIYLK (peptide IV, as shown in SEQ ID 04), WPRDPFTR (peptide V, as shown in SEQ ID 05), including its derivatives or its pharmaceutically acceptable salts. It should be noted that in this specification, the peptides composed of the amino acid sequences represented by the sequence numbers are also simply referred to as peptide 1, peptide 2, peptide 3, peptide 4, peptide 5, the same as peptide I, peptide II, peptide III, peptide IV, peptide V. Among the derivatives of the peptide of the present invention, the C-terminus of the peptide represented by a specific amino acid sequence can be any one of carboxyl (-COOH), carboxylate (-COO-), amide (-CONH2) or ester (-COOR). As R in the ester, for example, C1-6 chain alkyl such as methyl, ethyl, n-propyl, isopropyl or n-butyl can be cited; for example, C3-8 cycloalkyl such as cyclopentyl, cyclohexyl; for example, C6-12 aryl such as phenyl, α-naphthyl; for example, phenyl-C1-2 chain alkyl such as benzyl, phenethyl or α-naphthyl-C1-2 chain alkyl such as α-naphthylmethyl and other C7-14 aralkyl; in addition, pivaloyloxymethyl and the like widely used as oral esters can also be cited. As the amide form, amide can be cited; amide substituted by one or two C1-6 chain alkyls; amide substituted by one or two C1-6 chain alkyls substituted by phenyl; amide of a five-membered to seven-membered azacycloalkane in which two substituents together with the nitrogen atom to which they are bonded form a morpholino, piperidyl or the like containing the nitrogen atom of the amide group.

[0107] In an embodiment of the present invention, the peptide derivatives of the present invention further include: peptide derivatives in which the amino group at the N-terminus is protected by a protecting group (for example, C2-6 alkanoyl such as formyl, acetyl, etc., C1-6 acyl, etc.); peptide derivatives in which the glutamyl group generated by cleavage in vivo at the N-terminal side is pyroglutamylated; peptide derivatives in which substituents on the side chains of amino acids in the molecule (for example, -OH, -SH, amino group, imidazole group, indole group, guanidine group, etc.) are protected by appropriate protecting groups (for example, C2-6 alkanoyl such as formyl, acetyl, etc., C1-6 acyl, etc.).

[0108] In an embodiment of the present invention, the side chains of the amino acids constituting the peptide derivatives of the present invention can be modified with arbitrary substituents. The substituents are not particularly limited, and examples thereof include a fluorine atom, a chlorine atom, a cyano group, a hydroxyl group, a nitro group, an alkyl chain, a cycloalkyl group, an alkoxy group, an amino group, a phosphate group, etc. In addition, the substituents on the side chains can be protected by protecting groups. In this technical field, substituents, protecting groups, etc. have been well established in the past, and thus these groups can be used in the present invention.

[0109] In an embodiment of the present invention, the peptides of the present invention can form salts, and preferably physiologically acceptable salts as the salts. As physiologically acceptable salts, for example, salts with acids such as hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, palmitic acid, etc.; salts with alkali metals or alkaline earth metals such as sodium, potassium, calcium, or salts with hydroxides or carbonates of aluminum; salts with triethylamine, benzylamine, diethanolamine, tert-butylamine, dicyclohexylamine, arginine, etc.

[0110] In an embodiment of the present invention, as long as the peptides of the present invention maintain the characteristics of the original peptides, they can contain D-amino acids and can also contain non-natural amino acids. In addition, as long as the peptides or their derivatives of the present invention maintain the characteristics of the original peptides, other substances can be linked to the peptides. Examples of other substances that can be linked to the peptides include other peptides, lipids, sugars or sugar chains, acetyl groups, natural or synthetic polymers, etc. In addition, as long as the peptides of the present invention maintain the characteristics of the original peptides, they can also be modified by sugar chain addition, side chain oxidation, phosphorylation, etc. The serine of the peptides of the present invention can also be phosphorylated.

[0111] That is, the present invention includes compounds that are derivatives (or modified forms) of peptides 1-5 and exhibit the physiological activities of peptides 1-5 (the osteogenic effect or the effect of promoting fracture healing described later).

[0112] In an embodiment of the present invention, the peptide of the present invention can be easily prepared by solid-phase synthesis (Fmoc method, Boc method) or liquid-phase synthesis according to a generally known peptide synthesis protocol. In addition, it can be prepared using a transformant into which an expression vector is introduced, the expression vector containing DNA encoding the peptide, derivative, or salt thereof of the present invention. In addition, a peptide can be obtained using a transformant into which an expression vector is introduced, and the peptide can be prepared by cleaving the peptide using an appropriate protease or peptidase, the expression vector containing DNA encoding a peptide partially containing the peptide, derivative, or salt thereof of the present invention. In addition, it can be prepared by using methods such as in vitro transcription and translation. Since there are many known derivatives or modifiers for maintaining the physiological activity of peptides, the present invention can also be based on these substances.

[0113] In addition, the peptide of the present invention can be obtained by purifying the hydrolyzate of egg yolk protein. The method for preparing the hydrolyzate of egg yolk protein and the method for purifying the peptide are not particularly limited, and known methods can be appropriately selected. Specifically, for example, there can be mentioned: preparing defatted egg yolk powder, using an enzyme such as protease to prepare the hydrolyzate, and using various chromatographic methods such as ultrafiltration and HPLC (high performance liquid chromatography) to purify the target peptide.

[0114] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0115] Example 1 Effects of different pretreatment methods on the yield of hydrolyzed egg yolk polypeptide:

[0116] (1) To study the effect of the temperature of alkali heat treatment on the yield of hydrolyzed egg yolk polypeptide, a total of eight groups of experiments were set up in this example. For each group, 100 g of defatted egg yolk powder was taken and dissolved in 670 ml of water.

[0117] (2) For the first group, the pH of the liquid material was adjusted to 9.5, and water bath heating was carried out at 25 °C for 15 minutes.

[0118] For the second group, the pH of the liquid material was adjusted to 9.5, and water bath heating was carried out at 40 °C for 15 minutes.

[0119] For the third group, the pH of the liquid material was adjusted to 9.5, and water bath heating was carried out at 50 °C for 15 minutes.

[0120] For the fourth group, the pH of the liquid material was adjusted to 9.5, and water bath heating was carried out at 60 °C for 15 minutes.

[0121] For the fifth group, the pH of the liquid material was adjusted to 9.5, and water bath heating was carried out at 70 °C for 15 minutes.

[0122] For the sixth group, the pH of the liquid material was adjusted to 9.5, and water bath heating was carried out at 80 °C for 15 minutes.

[0123] The pH of the seventh group of feed liquid was adjusted to 9.5, and it was heated in a water bath at 90 °C for 15 minutes. The pH of the eighth group of feed liquid was adjusted to 9.5, and it was heated in a water bath at 90 °C for 15 minutes, and then wet grinding was carried out.

[0124] Specifically, wet grinding was carried out using a colloid mill. The emulsification fineness of the colloid mill was 2 μm, the motor power was 1.5 kw, and it passed through the colloid mill three times to obtain a defatted egg yolk powder homogenate.

[0125] (3) The temperature of the defatted egg yolk powder homogenate obtained in step (2) was adjusted to 55 °C, and 2 g of alkaline protease Alcalase was added respectively for enzymatic hydrolysis. The stirring enzymatic hydrolysis time was 4 h until the pH no longer decreased, and the reaction was regarded as terminated.

[0126] (4) After enzymatic hydrolysis, the supernatant was obtained by centrifugation at 5000 rpm for 25 min using a centrifuge;

[0127] (5) The obtained supernatant was spray-dried at an inlet air temperature of 180 °C and an outlet air temperature of 80 °C to obtain a hydrolyzed egg yolk powder product. The polypeptide yield was calculated by weighing the dry weight of the solid precipitate after centrifugation.

[0128] Egg yolk protein is prone to opening the protein structure at high temperatures, exposing the protease cleavage sites. There is a high content of phosvitin in egg yolk protein. Phosvitin is a highly phosphorylated protein, and most of the protease cleavage sites are wrapped inside the phosphate groups, resulting in a very low degree of hydrolysis of natural phosvitin by proteases. Adding strong base can partially dephosphorylate phosvitin, thereby exposing the protease cleavage sites. Furthermore, the alkaline protease added subsequently is an endonuclease, which can fully hydrolyze inside the protein with an open spatial structure, improving the enzymatic hydrolysis efficiency. As Figure 2 shown, the yield of egg yolk polypeptide is higher after pretreatment operations such as alkali-heat and wet grinding, and the yield is 60%. Moreover, in the actual operation process, the enzymatic hydrolysis efficiency of the seventh group is high, and the pH no longer decreases when the enzymatic hydrolysis time reaches 2.5 h.

[0129] Molecular weight detection of the hydrolyzed egg yolk powder product in Example 2

[0130] The hydrolyzed egg yolk powder product obtained in the eighth group of Example 1 was subjected to molecular weight detection. The detection results are shown in Table 1 and Figure 3 as shown. It can be seen from Table 1 and Figure 3 that: the molecular weights of all peptide segments of the hydrolyzed egg yolk powder are less than 250000 Da, and the molecular weight distribution of the main peptide segments (97%) is concentrated in the range of 180 - 2000 Da, indicating that the small peptides from dipeptides to pentapeptides have the highest content in the hydrolyzed egg yolk powder product. These small peptide segments are most easily absorbed by the human body, indicating that the hydrolyzed egg yolk powder product has good in vivo absorption performance and nutritional value.

[0131] Table 1 Molecular weight distribution of polypeptides in hydrolyzed egg yolk powder products

[0132] Retention time Mn Mw MP Area % Area 1 11.522 17794 22008 17777 352349 0.26 2 13.853 6473 6758 4999 188637 0.14 3 14.792 3575 3642 3000 857765 0.63 4 15.537 2355 2385 2000 2424432 1.79 5 16.812 1274 1321 999 14168380 10.49 6 18.083 657 684 500 34043021 25.20 7 19.459 280 304 237 74414291 55.08 8 19.957 128 139 180 8660747 6.41

[0133] After detection, the polypeptides in the obtained hydrolyzed egg yolk powder products are mainly composed of the amino acid sequences represented by FRTPPFGGF (peptide I, as shown in SEQ ID 01), HGFWPRDPFTPS (peptide II, as shown in SEQ ID 02), PFAEYPTYK (peptide III, as shown in SEQ ID 03), SRPILPIYLK (peptide IV, as shown in SEQ ID 04), and WPRDPFTR (peptide V, as shown in SEQ ID 05). It should be noted that in this specification, the peptides composed of the amino acid sequences represented by the sequence numbers are also simply referred to as peptide 1, peptide 2, peptide 3, peptide 4, and peptide 5, the same as peptide I, peptide II, peptide III, peptide IV, and peptide V.

[0134] Comparison of the enzymatic hydrolysis effects of different proteases in Example 3

[0135] A method for preparing hydrolyzed egg yolk powder, which is different from Example 1 in that the enzymatic hydrolysis treatment method is different. Specifically, on the basis of the alkaline protease hydrolysis in Example 1, neutral protease secondary hydrolysis is carried out again, including the following steps:

[0136] (1) Dissolve 100 g of defatted egg yolk powder in 670 ml of water;

[0137] (2) Adjust the pH of the feed liquid to 9.5, carry out heat treatment at 90 °C for 15 minutes, and use a colloid mill for wet grinding. The emulsification fineness of the colloid mill is 2 μm, the motor power is 1.5 kw, and pass through the colloid mill three times to obtain defatted egg yolk powder homogenate;

[0138] (3) Adjust the pH of the obtained defatted egg yolk powder homogenate to 9.5, adjust the temperature to 55 °C, add 2 g of alkaline protease Alcalase enzyme for enzymatic hydrolysis, and stir the enzymatic hydrolysis time for 4 h. When the pH no longer drops, it is regarded as the end of the reaction;

[0139] (4) Adjust the pH to 8.5, add 2 g of neutral protease (TF925S) and carry out the second enzymatic hydrolysis at 55 °C, stir the enzymatic hydrolysis time for 1 h, and when the pH no longer drops, it is regarded as the end of the reaction;

[0140] (5) After enzymatic hydrolysis, centrifuge at 5000 rpm for 25 min with a tubular centrifuge to obtain the supernatant;

[0141] (6) Carry out desalting treatment on the supernatant;

[0142] (7) Spray-dry the obtained supernatant, with an inlet air temperature of 180 °C and an outlet air temperature of 80 °C to obtain a hydrolyzed egg yolk powder product;

[0143] The obtained hydrolyzed egg yolk powder product was subjected to molecular weight detection, and the detection results are shown in Table 2 and Figure 4 as shown, from Table 2 and Figure 4 it can be seen that there is almost no difference between the hydrolyzed egg yolk powder obtained by adding neutral protease for the second enzymatic hydrolysis and the hydrolyzed egg yolk powder without adding neutral protease. This indicates that adding neutral protease for the second enzymatic hydrolysis has no effect on the hydrolyzed egg yolk powder product. The target hydrolyzed egg yolk powder product with the molecular weight distribution of the main peptide segments (97%) concentrated in the range of 180 - 2000 Da can be directly obtained by using alkaline protease for one-step enzymatic hydrolysis, and it mainly contains small peptides that are easily absorbed by the human body.

[0144] Table 2 Polypeptide molecular weight distribution in the hydrolyzed egg yolk powder product obtained by secondary enzymatic hydrolysis

[0145]

[0146]

[0147] Example 4 Study on the osteoblast precursor cell proliferation promoting activity of polypeptides

[0148] (1) Culturing cell suspension

[0149] Using α-MEM culture medium containing 10% FBS, mouse osteoblast-like cell line MC3T3-E1 was cultured until nearly confluent at 37 °C and 5% CO2 - 95% air, and then the cells were collected by trypsin treatment. The collected cells were suspended in the above α-MEM culture medium to prepare a cell suspension (1×10 5 cells / mL). This cell suspension was seeded on a 96-well plate at 100 μL per well and pre-cultured at 37 °C and 5% CO2 - 95% air.

[0150] (2) Preparing polypeptide solutions with a series of mass concentration gradients

[0151] Five synthesized polypeptides and the hydrolyzed egg yolk powder obtained in the eighth group of Example 1 were respectively prepared into polypeptide solutions with a series of mass concentration gradients of 1 μg / ml, 10 μg / ml, and 100 μg / ml.

[0152] (3) Continuous culture

[0153] After the cell suspension in step (1) was incubated at a constant temperature for 24 h, the original culture medium was removed, and the polypeptide solutions with a series of mass concentration gradients prepared in step (2) were added at 200 μL per well to the 96-well plate. Then, it was continuously cultured in a constant temperature incubator at 37 °C and a CO2 volume fraction of 5% for 48 h. The blank group was added with an equal volume of culture medium, and the proliferation rate was measured by the CCK-8 method. Each experiment was repeated 6 times in parallel. The relative proliferation rate of pre-osteoblasts was calculated according to the following formula.

[0154]

[0155] Wherein: A sample is the absorbance of the sample solution; A blank is the absorbance of the blank group solution.

[0156] The results are shown in Figure 5 , polypeptides 1-5 and hydrolyzed egg yolk powder both promoted the proliferation of osteoblast precursor cells, and the promotion effect was positively correlated with the mass concentration of the polypeptide solution; in particular, the promotion effect of the hydrolyzed egg yolk powder rich in polypeptides 1-5 was the most significant.

[0157] The above are only the preferred embodiments of the present invention. It should be pointed out that: for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A hydrolyzed egg yolk powder rich in polypeptide growth factor-like, characterized in that, The hydrolyzed egg yolk powder contains growth factor-like polypeptides, and the growth factor-like polypeptides include: Polypeptide I, with the amino acid sequence of FRTPPFGGF, Polypeptide II, with the amino acid sequence of HGFWPRDPFTPS, Polypeptide III, with the amino acid sequence of PFAEYPTYK, Polypeptide IV, with the amino acid sequence of SRPILPIYLK, Polypeptide V, with the amino acid sequence of WPRDPFTR.

2. The hydrolyzed egg yolk powder according to claim 1, wherein The molecular weight of all peptide segments of the hydrolyzed egg yolk powder is less than 250,000 Da.

3. The hydrolyzed egg yolk powder according to claim 2, wherein More than 97% of the peptide segments of the hydrolyzed egg yolk powder have a molecular weight distribution concentrated in the range of 180 - 2000 Da.

4. The hydrolyzed egg yolk powder according to claim 3, characterized in that, 97% - 98% of the peptide segments of the hydrolyzed egg yolk powder have a molecular weight distribution concentrated in the range of 180 - 2000 Da.

5. A method for preparing a hydrolyzed egg yolk powder rich in growth factor-like polypeptides as described in any one of claims 1-4, characterized in that, It includes the following steps: (1) Pretreatment: After dissolving the defatted egg yolk powder, under the conditions of pH 9 - 10, perform alkali heat treatment at 50°C - 90°C, and then perform wet grinding treatment to obtain a pretreated egg yolk powder solution; (2) Adjust the pretreated egg yolk powder solution obtained in step (1) to pH 9 - 10, then add alkaline protease, and perform enzymatic hydrolysis at 55°C ± 5°C to obtain an enzymatic hydrolysate; (3) Centrifuge the enzymatic hydrolysate obtained in step (2) to obtain a supernatant; (4) Perform nanofiltration desalination and concentration on the supernatant obtained in step (3) to obtain a concentrated solution; (5) Dry the concentrated solution obtained in step (4) to obtain the hydrolyzed egg yolk powder.

6. The preparation method according to claim 5, characterized in that, In step (1), the dissolution concentration of the defatted egg yolk powder is 6 - 10 ml of water / g of defatted egg yolk powder.

7. The preparation method according to claim 6, characterized in that, The dissolution concentration of the defatted egg yolk powder is 6 - 8 ml of water / g of defatted egg yolk powder.

8. The preparation method according to claim 5, characterized in that, In step (1), the time of the alkali heat treatment is 15 - 30 min.

9. The preparation method according to claim 8, characterized in that, The time of the alkali heat treatment is 15 - 20 min.

10. The preparation method according to claim 9, wherein The time of the alkali heat treatment is 15 min.

11. According to the preparation method described in claim 5, characterized in that, In step (1), the pH of the alkali heat treatment is 9 - 9.

5.

12. The preparation method according to claim 11, wherein, The pH of the alkali heat treatment is 9.

5.

13. The preparation method according to claim 5, characterized in that, In step (1), the temperature of the alkali heat treatment is 60°C - 90°C.

14. The preparation method according to claim 13, wherein, The temperature of the alkali heat treatment is 70°C - 90°C.

15. The preparation method according to claim 14, wherein The temperature of the alkali heat treatment is 80°C - 90°C.

16. The preparation method according to claim 15, characterized in that, The temperature of the alkali heat treatment is 90°C.

17. The preparation method according to claim 5, characterized in that, In step (1), the wet grinding treatment includes wet grinding using a colloid mill. The emulsification fineness of the colloid mill is 2 μm, the motor power is 1.5 kw, and the defatted egg yolk powder is passed through the colloid mill three times to obtain a defatted egg yolk powder homogenate.

18. The preparation method according to claim 5, wherein, In step (2), the alkaline protease is Alcalase enzyme.

19. The preparation method according to claim 18, wherein, The time of the Alcalase enzymatic hydrolysis is 2 - 4 h.

20. The preparation method according to claim 19, characterized in that, The time of the Alcalase enzyme enzymatic hydrolysis is 3 h.

21. The preparation method according to claim 18, characterized in that, The addition amount of the Alcalase enzyme alkaline protease is 1.5 - 2.5 wt% of the defatted egg yolk powder.

22. The preparation method according to claim 21, wherein The addition amount of the Alcalase enzyme alkaline protease is 2.0 wt% of the defatted egg yolk powder.

23. The preparation method according to claim 5, characterized in that, In step (3), the centrifugation includes centrifugation using a tubular centrifuge.

24. The preparation method according to claim 23, wherein The centrifugation rate is 3000 - 8000 rpm, and the centrifugation time is 15 - 35 min.

25. The preparation method according to claim 24, wherein, The centrifugation rate is 5000 rpm, and the centrifugation time is 25 min.

26. The preparation method according to claim 5, characterized in that, In step (4), the nanofiltration desalination and concentration includes passing the supernatant through a 150 Da membrane for nanofiltration desalination treatment.

27. The preparation method according to claim 5, wherein In step (5), the drying includes spray drying or freeze drying.

28. The preparation method according to claim 27, wherein, The spray drying includes an inlet air temperature of 180°C and an outlet air temperature of 80°C.

29. Use of the hydrolyzed egg yolk powder according to any one of claims 1-4, or the hydrolyzed egg yolk powder prepared by the method according to any one of claims 5-28, in the preparation of a medicament for promoting bone growth.

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