Hydrolyzed egg yolk powder rich in growth factor-like polypeptide as well as preparation method and application of hydrolyzed egg yolk powder

Abstract

The invention discloses hydrolyzed egg yolk powder rich in growth factor-like polypeptide and a preparation method and application thereof, relates to the technical field of biology and the field of nutrition and health, and particularly relates to the hydrolyzed egg yolk powder rich in growth factor-like polypeptide and application of the hydrolyzed egg yolk powder as a medicament for promoting bone growth. The hydrolyzed egg yolk powder is extracted from egg yolk through a specific enzymolysis technology, and is rich in growth factor-like polypeptides with a remarkable osteogenesis promoting function. The hydrolyzed egg yolk powder and the growth factor-like polypeptide can be used as food additives or medicine components, can effectively promote bone growth, and have remarkable effects on treating or preventing bone health related diseases such as osteogenesis imperfecta, osteoporosis and arthrosis deformans.

Description

Technical Field

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

[0002] With the accelerated pace of life and the intensification of the aging population, bone health issues are receiving increasing attention. Bone diseases such as osteogenesis imperfecta, osteoporosis and osteoarthritis seriously affect the quality of life of patients. At present, although there are a variety of treatment methods, the efficacy is not ideal, and there are problems such as large side effects and long treatment cycles. Therefore, the development of 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 a variety of bioactive substances. Among them, growth factor-like peptides, as an important class of bioactive substances, have the functions of promoting cell proliferation, differentiation and tissue repair, and have a significant promoting effect on bone growth. However, there are currently no hydrolyzed egg yolk powder products rich in growth factor-like peptides on the market. Summary of the invention

[0004] Purpose 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 and a preparation method and application thereof. The polypeptide of the present invention is derived from the hydrolyzed egg yolk powder and is extracted from the egg yolk powder by a specific enzymatic hydrolysis or hydrolysis technology. The polypeptide is rich in human epidermal growth factor-like activity, can simulate the effect of human epidermal growth factor (EGF), stimulate the proliferation and differentiation of osteoblasts, promote the synthesis and secretion of bone matrix, and thus accelerate bone growth and repair. At the same time, the polypeptide can also inhibit the activity of osteoclasts, reduce bone loss, and increase bone density and bone strength.

[0005] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is:

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

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

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

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

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

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

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

[0013] Polypeptide Ⅰ, amino acid sequence is FRTPPFGGF,

[0014] Polypeptide II, amino acid sequence is HGFWPRDPFTPS,

[0015] Polypeptide III, amino acid sequence is PFAEYPTYK,

[0016] Polypeptide IV, amino acid sequence is SRPILPIYLK,

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

[0018] Optionally, in some embodiments of the present invention, the molecular weight of all peptides in the hydrolyzed egg yolk powder is less than 250,000 Da.

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

[0020] Further 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 180-2000Da.

[0021] The third object of the present invention is to provide a method for preparing hydrolyzed egg yolk powder rich in growth factor-like polypeptides, characterized in that it comprises the following steps:

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

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

[0024] (3) centrifuging the enzymatic hydrolyzate obtained in step (2) to obtain a supernatant;

[0025] (4) performing nanofiltration desalination and concentration on the supernatant obtained in step (3) to obtain a concentrated solution;

[0026] (5) drying the concentrated solution obtained in step (4) to obtain 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 commercially available defatted egg yolk powder, or low-value defatted egg yolk powder obtained by industrial extraction of lecithin, with a protein content of 60%-90%.

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

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

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

[0032] Optionally, in some embodiments of the present invention, the pH of the alkaline heat treatment is 9-9.5, and more preferably 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] Further optionally, in some embodiments of the present invention, the temperature of the alkali heat treatment is 80°C-90°C, and more preferably 90°C.

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

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

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

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

[0040] Optionally, in some embodiments of the present invention, the added 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 amount of alkaline protease added is 1.5-2.5 wt % of defatted egg yolk powder, and more preferably 2.0 wt % of defatted egg yolk powder.

[0042] Optionally, in some embodiments of the present invention, in step (3), the centrifugation includes centrifugation in 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] Further 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 includes passing the supernatant through a 150Da membrane for nanofiltration desalination treatment.

[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 air inlet temperature of 180°C and an air outlet temperature of 80°C.

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

[0049] (1) adding water to dissolve defatted egg yolk powder to 6-8 ml water / g defatted egg yolk powder, adding sodium hydroxide to adjust the pH to 9.5, performing heat treatment at 90° C. for 15-30 min, and then performing wet grinding to obtain a defatted egg yolk powder homogenate;

[0050] (2) adjusting the obtained defatted egg yolk powder slurry to a pH of 9-10, and then adding alkaline protease for enzymatic hydrolysis;

[0051] (3) centrifuging after enzymatic hydrolysis to obtain a supernatant;

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

[0053] (5) spray-drying the final supernatant to obtain hydrolyzed egg yolk powder.

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

[0055] The fourth object of the present invention is to provide the use 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 food or medicine having the effect of promoting bone growth.

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

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

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

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

[0060] Polypeptide IV, 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 purified from the hydrolyzed egg yolk powder, or the polypeptide is synthesized separately.

[0063] The fifth object of the present invention is to provide the use of the above-mentioned human epidermal growth factor-like polypeptide in the preparation of food or medicine having 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 peptides, it has a significant bone-promoting function;

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

[0067] (3) It can be used as a food additive or drug ingredient, which is convenient and flexible;

[0068] (4) It has significant effects on the treatment or prevention of diseases related to bone health.

[0069] (5) The polypeptide of the present invention has a significant effect in promoting bone health. It can be used as an active ingredient in health foods, nutritional supplements or medicines to prevent and treat bone diseases such as osteoporosis and fractures, and promote bone health and growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1This is the spectrum of TIC&BPC of nano-HPLC-MS / MS detection results.

[0071] Figure 2 This is a graph showing the molecular weight of polypeptides in the hydrolyzed egg yolk powder product in Example 1.

[0072] Figure 3 This is a graph showing the molecular weight of polypeptides in the hydrolyzed egg yolk powder product in Example 2.

[0073] Figure 4 The present invention is a specific embodiment of the effect of polypeptides I-V on the cell proliferation rate of MC3T3-E1.

[0074] Figure 5 The proliferation promoting effect of polypeptides Ⅰ-Ⅴ and hydrolyzed egg yolk powder on osteoblast precursor cells. DETAILED DESCRIPTION

[0075] The present invention will be further described below in conjunction with the accompanying drawings and Examples. According to the following examples, the present invention can be better understood. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples 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 human epidermal growth factor-like activity, can simulate the effect 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, amino acid sequence is FRTPPFGGF,

[0078] Polypeptide II, amino acid sequence is HGFWPRDPFTPS,

[0079] Polypeptide III, amino acid sequence is PFAEYPTYK,

[0080] Polypeptide IV, amino acid sequence is SRPILPIYLK,

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

[0082] Another aspect of the present invention provides a hydrolyzed egg yolk powder rich in human epidermal growth factor analogs (growth factor-like polypeptides), which are rich in human epidermal growth factor-like activity, can simulate the effects 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, amino acid sequence is FRTPPFGGF,

[0084] Polypeptide II, amino acid sequence is HGFWPRDPFTPS,

[0085] Polypeptide III, amino acid sequence is PFAEYPTYK,

[0086] Polypeptide IV, amino acid sequence is SRPILPIYLK,

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

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

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

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

[0091] (3) centrifuging after enzymatic hydrolysis to obtain a supernatant;

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

[0093] (5) The final supernatant is spray-dried 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 obtained by industrial extraction of lecithin, with a protein content of 60%-90%.

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

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

[0097] The hydrolyzed egg yolk powder and polypeptide obtained by the invention can promote bone formation and can be used in preparing food and medicines capable of promoting bone growth.

[0098] In the embodiments of the present invention, the structural identification of the polypeptides is performed by nano-HPLC-MS / MS analysis, and the specific analysis method is as follows: ~200 ng of total peptides are taken from each sample and separated by the nano-UPLC liquid phase system Evosepone, and then combined with a mass spectrometer (timsTOFPro2) equipped with a nanoliter ion source for data acquisition. Chromatographic separation is performed using a 150μmID×15cm reverse phase chromatographic column (PePSepC18, 1.9μm, 150μm×15cm, Bruker, Germany). The mobile phase uses an acetonitrile-water-formic acid system, in which 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 balanced with 100% phase A, the sample is directly loaded onto the chromatographic column by the automatic sampler, and then separated by the chromatographic column gradient, with a gradient time of 44min.

[0099] In the embodiment of the present invention, the mass spectrometer adopts DDAPaSEF mode for DDA data acquisition, and the scanning range is from 100-1700 m / z. During the PASEFMS / MS scanning process, the collision energy increases linearly with the ion mobility, from 20 eV (1 / K0=0.6 Vs / cm2) to 59 eV (1 / K0=1.6 Vs / cm2). The original data file uses SpectroMine (4.2.230428.52329; Biognosys AG) software to apply the Pulsar search engine to perform database search, and qualitative analysis is performed after the search is completed. The TIC&BPC results of nano-HPLC-MS / MS are shown in Figure 1 Shown

[0100] In the embodiment of the present invention, the egg yolk polypeptide is screened by computer virtual screening to screen out peptides that may have bone growth promoting activity, and the specific process is as follows:

[0101] Molecular docking was performed on all peptides identified by mass spectrometry: human epidermal growth factor receptor (EGFR) was selected as the receptor protein, human epidermal growth factor (EGF) was selected as the original ligand, homology modeling was used to find the active center of EGFR, and AutoDockVina was used to perform molecular docking on EGFR and egg yolk protein peptides. They were arranged in ascending order of binding energy, and the first five peptides (binding energy <-7) were selected. According to the literature, the binding energy of AutoDockVina less than -7 is considered reliable.

[0102] In an embodiment of the present invention, the method for verifying the activity of the polypeptide in promoting osteoblast proliferation is as follows:

[0103] Mouse preosteoblast cell line MC3T3-E1 cells were inoculated in 96-well plates at 2×104 / mL and 100μL per well. After constant temperature culture for 24h, the original culture medium was removed, and then a series of peptide solutions with mass concentration gradients were prepared and added to the 96-well plates at 200μL per well. The cells were cultured continuously for 48h in a constant temperature incubator at 37°C and 5% CO2 volume fraction. The blank group was added with an equal volume of culture medium, and the proliferation rate was determined by CCK-8 method. Each group of experiments was performed 4 times in parallel. The relative proliferation rate of preosteoblasts was calculated according to the following formula.

[0104]

[0105] Where: Asample is the absorbance of the sample solution; Ablank is the absorbance of the blank solution.

[0106] In an embodiment of the present invention, the peptide of the present invention is a peptide composed of an amino acid sequence 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), including derivatives thereof or pharmacologically acceptable salts thereof. It should be noted that in this specification, the peptides composed of the amino acid sequences represented by the sequence numbers are also referred to as peptide 1, peptide 2, peptide 3, peptide 4, peptide 5, peptide I, peptide II, peptide III, peptide IV, peptide V, respectively. In the derivatives of the peptide of the present invention, the C-terminus of the peptide represented by the specific amino acid sequence can be any one of a carboxyl group (-COOH), a carboxylate group (-COO-), an amide (-CONH2) or an ester (-COOR). R in the ester includes, for example, C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl or n-butyl; C3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; C6-12 aryl groups such as phenyl and α-naphthyl; C7-14 aryl groups such as phenyl-C1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C1-2 alkyl groups such as α-naphthylmethyl; and pivaloyloxymethyl, which is widely used as an ester for oral use. Amide forms include amides; amides substituted with one or two C1-6 alkyl groups; amides substituted with one or two C1-6 alkyl groups substituted with phenyl; and amides of azacycloalkanes having five-membered to seven-membered rings such as morpholinyl and piperidinyl, which are formed by two substituents together with the nitrogen atom to which they are bonded, including the nitrogen atom of the amide group.

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

[0108] In an embodiment of the present invention, the side chain of the amino acid constituting the derivative of the peptide in the present invention may be modified by any substituent. The substituent is not particularly limited, and for example, fluorine atoms, chlorine atoms, cyano groups, hydroxyl groups, nitro groups, chain alkyl groups, cycloalkyl groups, alkoxy groups, amino groups, phosphate groups, etc. may be listed. In addition, the substituent of the side chain may be protected by a protecting group. In this technical field, substituents, protecting groups, etc. have been fully established in the past, so in the present invention, these groups may be used.

[0109] In an embodiment of the present invention, the peptide of the present invention can form a salt, and as the salt, a physiologically acceptable salt is preferred. Examples of physiologically acceptable salts include salts with 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 with aluminum hydroxide or carbonate; salts with triethylamine, benzylamine, diethanolamine, tert-butylamine, dicyclohexylamine, arginine, etc.

[0110] In an embodiment of the present invention, the peptide of the present invention may contain D-amino acids or non-natural amino acids as long as the characteristics of the original peptide are maintained. In addition, the peptide of the present invention or its derivatives may be connected to other substances on the peptide as long as the characteristics of the original peptide are maintained. As other substances that can be connected to the peptide, for example, other peptides, lipids, sugars or sugar chains, acetyl groups, natural or synthetic polymers, etc. can be listed. In addition, as long as the peptide of the present invention maintains the characteristics of the original peptide, the peptide may also be modified by sugar chain addition, side chain oxidation, phosphorylation, etc. The serine of the peptide of the present invention may also be phosphorylated.

[0111] That is, the present invention includes compounds which are derivatives (or modified forms) of peptide 1-5 and which exhibit the physiological activity of peptide 1-5 (the osteogenesis effect or fracture healing promoting effect 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 the known general peptide synthesis scheme. In addition, it can be prepared using a transformant introduced with an expression vector, the expression vector containing a DNA encoding the peptide of the present invention, its derivative or its salt. In addition, the peptide can be obtained using a transformant introduced with an expression vector, and the peptide can be prepared by cutting the peptide using an appropriate protease or peptidase. The expression vector contains a DNA encoding a peptide containing a portion of the peptide of the present invention, its derivative or its salt. In addition, it can be prepared by using an invitro (extracellular) transcription and translation method. In the past, it was known that there were many derivatives or modified bodies for maintaining the physiological activity of the peptide, so 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 a known method can be appropriately selected. Specifically, for example, methods include: preparing defatted egg yolk powder, preparing hydrolyzate using enzymes such as protease, and purifying the target peptide using various chromatography methods such as ultrafiltration and HPLC (high performance liquid chromatography).

[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 Effect of different pretreatment methods on the yield of hydrolyzed egg yolk polypeptides:

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

[0117] (2) The first group of feed liquid was adjusted to pH = 9.5 and heated in a water bath at 25°C for 15 minutes.

[0118] The second group of feed liquid was adjusted to pH = 9.5 and heated in a water bath at 40°C for 15 minutes.

[0119] The third group of liquid was adjusted to pH = 9.5 and heated in a water bath at 50° C. for 15 minutes.

[0120] The fourth group of feed liquid was adjusted to pH = 9.5 and heated in a water bath at 60° C. for 15 minutes.

[0121] The fifth group of feed liquid was adjusted to pH = 9.5 and heated in a water bath at 70° C. for 15 minutes.

[0122] The sixth group of feed liquid was adjusted to pH = 9.5 and heated in a water bath at 80° C. for 15 minutes.

[0123] The seventh group of liquid was adjusted to pH = 9.5, and heated in a water bath at 90°C for 15 minutes. The eighth group of liquid was adjusted to pH = 9.5, and heated in a water bath at 90°C for 15 minutes, and then wet crushed.

[0124] The wet grinding method specifically adopts colloid mill wet grinding, the emulsification fineness of the colloid mill is 2um, the motor power is 1.5kw, and the colloid mill is passed three times to obtain the defatted egg yolk powder slurry.

[0125] (3) The defatted egg yolk powder slurry obtained in step (2) was adjusted to 55° C., and 2 g of alkaline protease Alcalase was added for enzymolysis. When the pH value no longer decreased after stirring for 4 h, the reaction was considered to be terminated.

[0126] (4) After enzymatic hydrolysis, centrifuge at 5000 rpm for 25 min to obtain the supernatant;

[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 easily opens the protein structure at high temperature, exposing the protease hydrolysis site. Egg yolk protein contains a lot of phosphoprotein, which is a highly phosphorylated protein. Most of the protease action sites are wrapped inside the phosphate group, resulting in a very low degree of protease hydrolysis of natural egg yolk phosphoprotein. Strengthening the alkali can partially dephosphorylate egg yolk phosphoprotein, thereby exposing the protease action site. The alkaline protease added later is an endonuclease, which can fully hydrolyze the protein inside the opened spatial structure and improve the efficiency of enzymatic hydrolysis. Figure 2 As shown, the yield of egg yolk polypeptide after alkaline heat and wet crushing pretreatment is higher, the yield is 60%, and in the actual operation process, the seventh group has high enzymatic hydrolysis efficiency, and the pH no longer decreases when the enzymatic hydrolysis time reaches 2.5h.

[0129] Example 2 Molecular Weight Detection of Hydrolyzed Egg Yolk Powder Products

[0130] The hydrolyzed egg yolk powder product obtained in Group 8 of Example 1 was tested for molecular weight. The test results are shown in Tables 1 and Figure 3 As shown in Table 1 and Figure 3 It can be seen that the molecular weight of all peptides in the hydrolyzed egg yolk powder is less than 250,000 Da, and the molecular weight distribution of the main peptides (97%) is concentrated in the range of 180-2000 Da, indicating that the highest content in the hydrolyzed egg yolk powder product is small peptides from dipeptides to pentapeptides, and these small peptides 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]

[0133]

[0134] After testing, the polypeptides in the obtained hydrolyzed egg yolk powder product 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 referred to as peptide 1, peptide 2, peptide 3, peptide 4, peptide 5, peptide I, peptide II, peptide III, peptide IV, peptide V, respectively.

[0135] Example 3 Comparison of enzymatic hydrolysis effects of different proteases

[0136] A method for preparing hydrolyzed egg yolk powder, which differs from Example 1 in that the enzymatic hydrolysis method is different. Specifically, based on the alkaline protease enzymatic hydrolysis in Example 1, a secondary enzymatic hydrolysis with a neutral protease is performed again, comprising the following steps:

[0137] (1) Dissolve 100g defatted egg yolk powder in 670ml water;

[0138] (2) adjusting the pH of the feed liquid to 9.5, heat-treating the feed liquid at 90° C. for 15 minutes, and grinding the feed liquid by wet method using a colloid mill with an emulsification fineness of 2 μm and a motor power of 1.5 kW. The feed liquid was passed through the colloid mill three times to obtain a defatted egg yolk powder slurry;

[0139] (3) The pH of the obtained defatted egg yolk powder slurry was adjusted to 9.5, the temperature was adjusted to 55° C., 2 g of alkaline protease Alcalase was added for enzymolysis, and the enzymolysis time was stirred for 4 h. When the pH no longer decreased, the reaction was considered to be terminated;

[0140] (4) Adjust the pH to 8.5, add 2 g of neutral protease (TF925S) and perform the second enzymolysis at 55 °C. Stir the enzymolysis for 1 h. The reaction is considered terminated when the pH no longer decreases.

[0141] (5) After enzymatic hydrolysis, the supernatant was obtained by centrifugation at 5000 rpm for 25 min in a tubular centrifuge;

[0142] (6) desalting the supernatant;

[0143] (7) spray drying the obtained supernatant at an inlet air temperature of 180° C. and an outlet air temperature of 80° C. to obtain a hydrolyzed egg yolk powder product;

[0144] The obtained hydrolyzed egg yolk powder product was subjected to molecular weight testing. The test results are shown in Table 2 and Figure 4 As shown in 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 shows that adding neutral protease for the second enzymatic hydrolysis has no effect on the hydrolyzed egg yolk powder product, and directly using alkaline protease for the first enzymatic hydrolysis can obtain the target hydrolyzed egg yolk powder product with the main peptides (97%) concentrated in the molecular weight range of 180-2000Da, which is mainly rich in small peptides that are easily absorbed by the human body.

[0145] Table 2 Molecular weight distribution of polypeptides in the hydrolyzed egg yolk powder product obtained by secondary enzymatic hydrolysis

[0146] Retention time Mn M MP area %area 1 11.514 18533 23239 17856 393561 0.35 2 13.853 6782 7085 4999 127307 0.11 3 14.792 3583 3651 3000 376382 0.33 4 15.537 2348 2379 2000 1071089 0.94 5 16.812 1282 1328 999 6963603 6.13 6 18.083 650 676 500 25529956 22.47 7 19.479 276 299 234 70629867 62.16 8 19.957 130 141 180 8527599 7.51

[0147] Example 4 Study on osteoblast precursor cell proliferation promoting activity using polypeptide

[0148] (1) Cultivated cell suspension

[0149] The cells were cultured in α-MEM medium containing 10% FBS at 37°C and 5% CO 2 Mouse osteoblast-like cell line MC3T3-E1 was cultured to near confluence under -95% air, and then the cells were collected by trypsin treatment. The collected cells were suspended in the above-mentioned α-MEM culture medium to prepare a cell suspension (1×10 5 The cell suspension was seeded in each well of a 96-well plate at 100 μL and incubated at 37°C and 5% CO 2 Pre-culture was performed under -95% air.

[0150] (2) Prepare a series of peptide solutions with a mass concentration gradient of

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

[0152] (3) Continuous culture

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

[0154]

[0155] Where: Asample is the absorbance of the sample solution; Ablank is the absorbance of the blank solution.

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

[0157] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A growth factor-like polypeptide, characterized in that The growth factor-like polypeptide comprises at least one of the following: Polypeptide Ⅰ, amino acid sequence is FRTPPFGGF, Polypeptide II, amino acid sequence is HGFWPRDPFTPS, Polypeptide III, amino acid sequence is PFAEYPTYK, Polypeptide IV, amino acid sequence is SRPILPIYLK, Polypeptide V, the amino acid sequence is WPRDPFTR.

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

3. The hydrolyzed egg yolk powder according to claim 2, characterized in that: The molecular weight of all peptide segments of the hydrolyzed egg yolk powder is less than 250,000 Da, and / or Preferably, more than 97% of the peptides in the hydrolyzed egg yolk powder have a molecular weight distribution of 180-2000 Da, and / or Preferably, the molecular weight distribution of 97%-98% of the peptide segments of the hydrolyzed egg yolk powder is concentrated in the range of 180-2000Da.

4. A method for preparing hydrolyzed egg yolk powder rich in growth factor-like polypeptides, characterized in that: The steps include: (1) Pretreatment: After the defatted egg yolk powder is dissolved, an alkali heat treatment is performed at 50°C-90°C under a pH of 9-10, and then a wet pulverization treatment is performed to obtain a pretreated egg yolk powder solution; (2) The pretreated egg yolk powder solution obtained in step (1) is adjusted to a pH of 9-10, and then alkaline protease is added to perform enzymolysis at 55°C ± 5°C to obtain an enzymolysis solution; (3) centrifuging the enzymatic hydrolyzate obtained in step (2) to obtain a supernatant; (4) performing nanofiltration desalination and concentration on the supernatant obtained in step (3) to obtain a concentrated solution; (5) drying the concentrated solution obtained in step (4) to obtain hydrolyzed egg yolk powder.

5. The preparation method according to claim 4, characterized in that: In step (1), the defatted egg yolk powder is dissolved in a concentration of 6-10 ml water / g defatted egg yolk powder, preferably 6-8 ml water / g defatted egg yolk powder, and / or Preferably, the alkali heat treatment time is 15-30 min, and / or Preferably, the alkali heat treatment time is 15-20 min, preferably 15 min, and / or Preferably, the pH of the alkaline heat treatment is 9-9.5, preferably 9.5, and / or Preferably, the temperature of the alkali heat treatment is 60°C-90°C, and / or Preferably, the temperature of the alkali heat treatment is 70°C-90°C, and / or Preferably, the temperature of the alkali heat treatment is 80°C-90°C, preferably 90°C.

6. The preparation method according to claim 4, characterized in that: In step (1), the wet pulverization treatment includes wet pulverization by a colloid mill, the emulsification fineness of the colloid mill is 2um, the motor power is 1.5kw, and the defatted egg yolk powder slurry is obtained by passing through the colloid mill three times.

7. The preparation method according to claim 4, characterized in that: In step (2), the alkaline protease includes Alcalase or trypsin, Preferably, the Alcalase enzymolysis time is 2-4h, preferably 3h, and / or Preferably, the trypsin hydrolysis time is 3-5h, preferably 4h, and / or Preferably, the amount of Alcalase enzyme alkaline protease added is 1.5-2.5wt% defatted egg yolk powder, preferably 2.0wt% defatted egg yolk powder, and / or Preferably, the addition amount of the trypsin alkaline protease is 1-3wt% defatted egg yolk powder, preferably 2.0wt% defatted egg yolk powder.

8. The preparation method according to claim 4, characterized in that: In step (3), the centrifugation includes centrifugation in a tubular centrifuge, and / or Preferably, the centrifugal speed is 3000-8000 rpm, the centrifugal time is 15-35 min, and / or Preferably, the centrifugal speed is 5000 rpm and the centrifugal time is 25 min.

9. The preparation method according to claim 4, characterized in that: In step (4), the nanofiltration desalination and concentration comprises passing the supernatant through a 150Da membrane for nanofiltration desalination treatment. In step (5), the drying comprises spray drying or freeze drying. Preferably, the spray drying includes an air inlet temperature of 180°C and an air outlet temperature of 80°C.

10. Use of the growth factor-like polypeptide according to claim 1, or the hydrolyzed egg yolk powder according to claim 2 or 3, or the hydrolyzed egg yolk powder prepared by the method according to any one of claims 3 to 9 in the preparation of food or medicine having the effect of promoting bone growth.

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