Preparation method of egg yolk active peptide for promoting proliferation of hair follicle dermal papilla cells, product and application thereof

Abstract

The present application discloses a preparation method of an egg yolk active peptide that promotes the proliferation of hair follicle dermal papilla cells, its product and application, and belongs to the field of egg yolk active peptide technology. The preparation method of the present application comprises: providing an egg yolk homogenized aqueous solution containing sulfite, and subjecting the egg yolk homogenized aqueous solution to ultrasonic shearing treatment, adding a composite protease containing neutral protease, alkaline protease and fermented chymosin to the obtained pretreated substrate solution for composite enzymatic hydrolysis treatment, followed by centrifugal stratification separation, removing the lower layer and vacuum freeze-drying to obtain an egg yolk active peptide that promotes the proliferation of hair follicle dermal papilla cells. The preparation method of the present application prepares an egg yolk active peptide with a new biological function, which has the function of promoting the proliferation of human dermal hair papilla cells and the secretion of vascular endothelial growth factor VEGF, and can significantly accelerate the growth rate of mammalian hair and increase the density of hair.

Description

Technical Field

[0001] The present application belongs to the technical field of egg yolk active peptides, and in particular relates to a preparation method of an egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells, and its product and application. Background Art

[0002] Egg yolk active peptides are peptide substances formed after the release of egg yolk protein. They have functional activities such as antioxidant, antibacterial and anti-inflammatory, immunity enhancement and blood pressure / blood sugar regulation. They can be used in food, health products or topical medicines to play an important role in biological health care and disease prevention.

[0003] Proteolysis is currently the most common method for preparing egg yolk active peptides. For example, He Chen et al. used Alcalase 2.4L to hydrolyze egg yolk powder twice to prepare egg yolk peptides that can bind to calcium ions and inhibit calcium phosphate precipitation, showing the potential ability to promote intestinal calcium absorption; the patent application CN 115537442 A discloses an efficient enrichment and preparation method for egg yolk phosphopeptides, which uses pancreatic enzymes or alkaline proteases to enzymatically delipidate egg yolks to prepare egg yolk phosphopeptides that promote bone growth.

[0004] However, although a variety of egg yolk active peptides have been widely studied and achieved significant results, there is still great potential for the development of egg yolk active peptides. In addition, the existing methods for preparing egg yolk active peptides generally have a relatively low yield, making it difficult to introduce industrial-scale production. Summary of the invention

[0005] The present application discloses a preparation method of an egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells, its product and application, thereby achieving the application of the egg yolk active peptide in promoting the proliferation of hair follicle dermal papilla cells or stimulating mammalian hair growth in vitro while effectively improving the product yield.

[0006] To achieve the above objectives, the present application provides in a first aspect a method for preparing an egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells, the preparation method comprising:

[0007] providing an egg yolk homogenate aqueous solution containing sulfite;

[0008] Performing ultrasonic shearing treatment on the egg yolk homogenous aqueous solution to obtain a pre-treated substrate solution;

[0009] Adding a composite protease to the pretreated substrate solution for composite enzymolysis treatment to obtain an enzymolysis product solution, wherein the composite protease contains neutral protease, alkaline protease and fermentation-type chymotrypsin;

[0010] After the enzyme is inactivated, the enzymatic hydrolysis product solution is subjected to centrifugal separation and the lower layer is taken out and vacuum freeze-dried to obtain the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells.

[0011] In some embodiments, the egg yolk homogenized aqueous solution is obtained by mixing egg yolk stock solution and water at a mass ratio of 1:4 and homogenizing;

[0012] The mass percentage of the sulfite in the egg yolk homogenous aqueous solution is 5%.

[0013] In some embodiments, when the ultrasonic shearing treatment is performed, the ultrasonic frequency is 20 kHz, the ultrasonic power is 420 W, and the ultrasonic time is 10 min.

[0014] In some embodiments, the mass ratio of neutral protease, alkaline protease and fermentation-type chymotrypsin contained in the composite protease is (0.5-1):(1-2):(0.1-0.5).

[0015] In some embodiments, the added amount of the composite protease is 15-35 g / 1000 g.

[0016] In some embodiments, when the composite enzymatic hydrolysis treatment is performed, the temperature is maintained at 65-75° C., the pH value is maintained at 7-10, and the time is 1.5-3 hours.

[0017] The second aspect of the present application provides an egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells, which is prepared by the preparation method described in the first aspect.

[0018] The third aspect of the present application provides the use of the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells in the present application for stimulating hair growth in mammals in vitro.

[0019] In some application schemes, the effective concentration of the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells is 40-160 μg / mL.

[0020] In some application schemes, the effective concentration of the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells is 80 μg / mL.

[0021] Compared with the prior art, the advantages or beneficial effects of the embodiments of the present application include at least:

[0022] The preparation method provided in the present application is to subject the homogenized aqueous solution of egg yolk to ultrasonic shearing in the presence of sulfite, and then subject it to a composite enzymatic hydrolysis treatment with a specific composite protease, thereby preparing an egg yolk active peptide with a new biological function, and the product yield can reach up to 70.05%. Among them, the physical and chemical index test shows that the egg yolk active peptide prepared in the present application has a relatively small molecular weight, specifically, the relative molecular weight within 1000Da accounts for 90.15%, and the relative molecular weight within 2000Da accounts for 96.6%, and the degree of hydrolysis can reach up to 45%, and the free amino acid accounts for up to 10.05%. It has good water solubility, clear solution, no fishy smell and low bitterness. More importantly, in vitro activity experiments have found that the egg yolk active peptide has the effect of promoting the proliferation of human dermal hair papilla cells and the secretion of vascular endothelial growth factor (VEGF), which can significantly accelerate the growth rate of mammalian hair and increase the density of hair. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The images of hair growth on the back of experimental mice provided in the examples of the present application at different days are taken;

[0025] Figure 2 HE staining of the experimental mouse back skin tissue section provided in the examples of the present application;

[0026] Figure 3 The example of the present application provides the change in the relative activity of HFDPC cells with the concentration of egg yolk active peptide AY1. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0028] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0029] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, and a, b, c can be single or multiple, respectively.

[0030] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0031] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0032] Those skilled in the art will appreciate that the numerical ranges in the embodiments of the present application are to be understood as each intermediate value between the upper and lower limits of the scope also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the scope.

[0033] Unless otherwise specified, the technical / scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs. Although this application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] In a first aspect, the present invention provides a method for preparing an egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells, preferably comprising the following steps:

[0035] providing an egg yolk homogenate aqueous solution containing sulfite;

[0036] Performing ultrasonic shearing treatment on the egg yolk homogenous aqueous solution to obtain a pre-treated substrate solution;

[0037] Adding a composite protease to the pretreated substrate solution for composite enzymolysis treatment to obtain an enzymolysis product solution, wherein the composite protease contains neutral protease, alkaline protease and fermentation-type chymotrypsin;

[0038] After the enzyme is inactivated, the enzymatic hydrolysis product solution is subjected to centrifugal separation and the lower layer is taken out and vacuum freeze-dried to obtain the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells.

[0039] It should be noted that the sulfite refers to a compound containing SO3 2- / or HSO3 - The water-soluble salt may preferably be a sulfite well known in the field of food additives, such as Na2SO3, K2SO3, NaHSO3 or KHSO3, etc., which is not particularly limited in the embodiments of the present application.

[0040] The present application example prepares egg yolk active peptides with new biological functions by subjecting the homogenized egg yolk aqueous solution to ultrasonic shearing in the presence of sulfite, and then subjecting it to a composite enzymatic treatment with a specific composite protease, and the product yield can reach up to 70.05%. Among them, the physical and chemical index tests show that the egg yolk active peptides prepared in this application have a relatively small molecular weight, specifically, the relative molecular weight within 1000Da accounts for 90.15%, the relative molecular weight within 2000Da accounts for 96.6%, the degree of hydrolysis can reach up to 45%, the free amino acid accounts for up to 10.05%, the water solubility is good, the solution is clear, there is no fishy smell and the bitterness is low. More importantly, the in vitro activity experiment found that the egg yolk active peptide has the effect of promoting the proliferation of human dermal hair papilla cells and the secretion of vascular endothelial growth factor (VEGF), which can significantly accelerate the growth rate of mammalian hair and increase the density of hair.

[0041] In a specific embodiment, the egg yolk homogenized aqueous solution is preferably obtained by mixing egg yolk stock solution and water at a mass ratio of 1:4 and homogenizing, and the mass percentage of the sulfite in the egg yolk homogenized aqueous solution is preferably 5%. The egg yolk stock solution refers to the fresh egg yolk liquid separated from the egg white.

[0042] It should be noted that, in the embodiment of the present application, after adding water to the egg yolk stock solution and homogenizing it, sulfite is added and its mass proportion is controlled. Firstly, it is beneficial to control the temperature of the egg yolk stock solution to avoid inactivation of protein due to excessive local temperature; secondly, it ensures that the sulfite is in full contact with the egg yolk stock solution, so that the egg yolk protein structure undergoes a chemical conformational change, making the protein structure tend to be loose and better exposing the hydrophobic groups, which is not only beneficial to the separation and precipitation of protein and oil, but also beneficial to increase the solubility of protein, thereby effectively increasing the degree of enzymatic hydrolysis of the enzymatic reaction, and facilitating the formation of peptide active substances with specific physicochemical properties and biological activity.

[0043] In a specific embodiment, when performing the ultrasonic shearing treatment, the ultrasonic frequency is preferably 20 kHz, the ultrasonic power is preferably 420 W, and the ultrasonic time is preferably 10 min.

[0044] It should be noted that the embodiments of the present application perform ultrasonic shearing treatment on the homogenized aqueous solution of egg yolk containing sulfite and control the ultrasonic conditions, so that the physical conformational change of the egg yolk protein structure can be achieved, which is beneficial to increase the solubility of the protein, thereby effectively increasing the degree of enzymatic hydrolysis of the enzymatic reaction; and the physical conformational change can be coupled with the chemical conformational transformation, which can further intensify the degree of enzymatic hydrolysis of the enzymatic reaction, making it easier to form peptide active substances with specific physicochemical properties and biological activity.

[0045] In a specific embodiment, the mass ratio of the neutral protease, alkaline protease and fermentation type chymotrypsin contained in the composite protease is preferably (0.5-1):(1-2):(0.1-0.5). Among them, the mass ratio of the neutral protease, alkaline protease and fermentation type chymotrypsin is more preferably (1:2:0.2) in addition to the endpoint values ​​(0.5:1:0.1) and (1:2:0.5).

[0046] It should be noted that the neutral protease is derived from Bacillus subtilis COROLASE 7089; the alkaline protease is derived from Bacillus licheniformis Alkaline Protease 2709; and the fermentation type chymosin is fermented from non-transgenic bacteria Bacillus licheniformis. Of course, the specific source of each protease is not limited in the present embodiment, and it is preferably obtained through commercial channels.

[0047] It should be noted that the embodiments of the present application select the specific composite protease described above and control its combination ratio, so as to achieve a more thorough enzymatic hydrolysis of egg yolk protein based on different enzymatic cleavage directions, thereby obtaining egg yolk active peptides with smaller molecular weight and more free amino acids. The small molecular weight and rich free amino acids are beneficial to enhancing the biological activity of egg yolk peptides.

[0048] In a specific embodiment, the added amount of the composite protease is preferably 15-35 g / 1000 g, wherein the added amount is more preferably 32 g / 1000 g in addition to the endpoint values ​​of 15 g / 1000 g and 35 g / 1000 g.

[0049] In a specific embodiment, when the composite enzymatic hydrolysis treatment is performed, the temperature is maintained between 65 and 75° C., the pH value is maintained between 7 and 10, and the time is 1.5 to 3 hours.

[0050] It should be noted that the temperature and pH value can be controlled to be constant or fluctuate within the range. The present application embodiment controls the enzymolysis conditions described above, which can effectively maintain the biological activity of different enzymes, facilitate the rapid progress of the enzymolysis reaction and form a greater degree of enzymolysis.

[0051] On the second aspect, the embodiments of the present application also provide an egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells prepared according to the method described above. Among them, the preparation method based on the embodiments of the present application can effectively improve the degree of enzymatic hydrolysis, improve the separation and precipitation rate of oil, increase the solubility of protein and release the number of free amino acids, so that the prepared egg yolk active peptide has a small molecular weight, rich free amino acids, and good water solubility. These physicochemical properties give the egg yolk active peptide the effect of promoting the proliferation of human dermal papilla cells and the secretion of vascular endothelial growth factor (VEGF), which can significantly accelerate the growth rate of mammalian hair and increase the density of hair.

[0052] On the third aspect, the embodiments of the present application provide the use of the egg yolk active peptides for promoting the proliferation of hair follicle dermal papilla cells according to the embodiments of the present application for in vitro stimulation of mammalian hair growth. Among them, based on the embodiments of the present application, it has the effect of promoting the proliferation of human dermal papilla cells and the secretion of vascular endothelial growth factor (VEGF), so after being used to stimulate mammalian hair growth in vitro, it can significantly accelerate the growth rate of mammalian hair and increase the density of hair, and transdermal administration is more convenient and safer than oral administration. It should be noted that based on the embodiments of the present application, the egg yolk active peptides have the effect of promoting the proliferation of hair follicle dermal papilla cells, and can therefore be used to prepare, including but not limited to, hair lotions.

[0053] The technical solution of the present application will be further described below in conjunction with specific embodiments. Example 1

[0054] This embodiment provides a method for preparing egg yolk active peptide AY1 that promotes the proliferation of hair follicle dermal papilla cells, and the steps are as follows:

[0055] Step 1: After peeling the fresh eggs and separating the egg yolk stock solution, take 200g of the egg yolk stock solution and add 800g of deionized water for homogenization, then add 5g of sodium sulfite to the obtained egg yolk homogenous aqueous solution to obtain an egg yolk homogenous aqueous solution containing sodium sulfite;

[0056] Step 2: ultrasonically treating the egg yolk homogenized aqueous solution containing sodium sulfite at an ultrasonic frequency of 20 kHz and an ultrasonic power of 420 W for 10 minutes to obtain a pretreated substrate solution;

[0057] Step 3: After adding composite protease at 32 g / 1000 g to the pretreated substrate solution, the temperature of the solution system was controlled to be 70° C. and the pH value was 9.8, and composite enzymatic hydrolysis was performed for 2 h to obtain an enzymatic hydrolysis product solution E1, wherein the composite protease is composed of neutral protease, alkaline protease and fermentation-type chymosin in a mass ratio of 1:2:0.2;

[0058] Step 4: After the enzymatic hydrolyzate solution E1 is inactivated at 100°C, the solution is separated by centrifugation (8000 rpm, 15 min), and the lower layer is removed and vacuum freeze-dried to obtain the egg yolk active peptide AY1 that promotes the proliferation of hair follicle dermal papilla cells. Example 2

[0059] This embodiment provides a method for preparing egg yolk active peptide AY2 that promotes the proliferation of hair follicle dermal papilla cells, and the steps are as follows:

[0060] Step 1: After peeling the fresh eggs and separating the egg yolk stock solution, take 200g of the egg yolk stock solution and add 800g of deionized water for homogenization, then add 5g of sodium sulfite to the obtained egg yolk homogenous aqueous solution to obtain an egg yolk homogenous aqueous solution containing sodium sulfite;

[0061] Step 2: ultrasonically treating the egg yolk homogenized aqueous solution containing sodium sulfite at an ultrasonic frequency of 20 kHz and an ultrasonic power of 420 W for 10 minutes to obtain a pretreated substrate solution;

[0062] Step 3: After adding composite protease at 32 g / 1000 g to the pretreated substrate solution, the temperature of the solution system was controlled to be 70° C. and the pH value was 9.8, and composite enzymatic hydrolysis was performed for 2 h to obtain an enzymatic hydrolysis product solution E2, wherein the composite protease is composed of neutral protease, alkaline protease and fermentation-type chymosin in a mass ratio of 0.5:1:0.1;

[0063] Step 4: After the enzymatic hydrolyzate solution E2 is inactivated at 100°C, the solution is separated by centrifugation (8000 rpm, 15 min), the lower layer is removed and vacuum freeze-dried to obtain the egg yolk active peptide AY2 that promotes the proliferation of hair follicle dermal papilla cells. Example 3

[0064] This embodiment provides a method for preparing egg yolk active peptide AY3 that promotes the proliferation of hair follicle dermal papilla cells, and the steps are as follows:

[0065] Step 1: After peeling the fresh eggs and separating the egg yolk stock solution, take 200g of the egg yolk stock solution and add 800g of deionized water for homogenization, then add 5g of sodium sulfite to the obtained egg yolk homogenous aqueous solution to obtain an egg yolk homogenous aqueous solution containing sodium sulfite;

[0066] Step 2: subjecting the egg yolk homogenized aqueous solution containing sodium sulfite to ultrasonic treatment for 10 minutes at an ultrasonic frequency of 20 kHz and an ultrasonic power of 420 W to obtain a pretreated substrate solution;

[0067] Step 3: After adding the composite protease at 32 g / 1000 g to the pretreated substrate solution, the temperature of the solution system was controlled to be 70° C. and the pH value was 9.8, and the composite enzymatic hydrolysis treatment was performed for 2 h to obtain an enzymatic hydrolysis product solution E3, wherein the composite protease is composed of neutral protease, alkaline protease and fermentation-type chymosin in a mass ratio of 1:2:0.5;

[0068] Step 4: After the enzymatic hydrolysis product solution E3 is inactivated at 100°C, the solution is separated by centrifugation (8000rpm, 15min), the lower layer is removed and vacuum freeze-dried to obtain the egg yolk active peptide AY3 that promotes the proliferation of hair follicle dermal papilla cells. Example 4

[0069] This embodiment provides a method for preparing egg yolk active peptide AY4 that promotes the proliferation of hair follicle dermal papilla cells, and the steps are as follows:

[0070] Step 1: After peeling the fresh eggs and separating the egg yolk stock solution, take 200g of the egg yolk stock solution and add 800g of deionized water for homogenization, then add 5g of sodium sulfite to the obtained egg yolk homogenous aqueous solution to obtain an egg yolk homogenous aqueous solution containing sodium sulfite;

[0071] Step 2: subjecting the egg yolk homogenized aqueous solution containing sodium sulfite to ultrasonic treatment for 10 minutes at an ultrasonic frequency of 20 kHz and an ultrasonic power of 420 W to obtain a pretreated substrate solution;

[0072] Step 3: After adding composite protease at 15 g / 1000 g to the pretreated substrate solution, the temperature of the solution system was controlled to be 65° C. and the pH value was 7, and composite enzymatic hydrolysis was performed for 3 h to obtain an enzymatic hydrolysis product solution E4, wherein the composite protease was composed of neutral protease, alkaline protease and fermentation-type chymosin in a mass ratio of 1:2:0.2;

[0073] Step 4: After the enzymatic hydrolyzate solution E4 is inactivated at 100°C, the solution is separated by centrifugation (8000 rpm, 15 min), the lower layer is removed and vacuum freeze-dried to obtain the egg yolk active peptide AY4 that promotes the proliferation of hair follicle dermal papilla cells. Example 5

[0074] This embodiment provides a method for preparing egg yolk active peptide AY5 that promotes the proliferation of hair follicle dermal papilla cells, and the steps are as follows:

[0075] Step 1: After peeling the fresh eggs and separating the egg yolk stock solution, take 200g of the egg yolk stock solution and add 800g of deionized water for homogenization, then add 5g of sodium sulfite to the obtained egg yolk homogenous aqueous solution to obtain an egg yolk homogenous aqueous solution containing sodium sulfite;

[0076] Step 2: subjecting the egg yolk homogenized aqueous solution containing sodium sulfite to ultrasonic treatment for 10 minutes at an ultrasonic frequency of 20 kHz and an ultrasonic power of 420 W to obtain a pretreated substrate solution;

[0077] Step 3: After adding composite protease at 35 g / 1000 g to the pretreated substrate solution, the temperature of the solution system was controlled to be 75° C. and the pH value was 7, and composite enzymatic hydrolysis was performed for 1.5 h to obtain an enzymatic hydrolysis product solution E5, wherein the composite protease is composed of neutral protease, alkaline protease and fermentation-type chymosin in a mass ratio of 1:2:0.2;

[0078] Step 4: After the enzymatic hydrolysis product solution E5 is inactivated at 100°C, the solution is centrifuged and separated into layers, and the lower layer is removed and vacuum freeze-dried to obtain the egg yolk active peptide AY5 that promotes the proliferation of hair follicle dermal papilla cells.

[0079] In order to illustrate the technical effect of the method described above, the present application provides comparative examples 1 to 4.

[0080] Comparative Example 1

[0081] Compared with Example 1, sodium sulfite is not added to the egg yolk homogenized aqueous solution in this comparative example, and the remaining steps are the same as those in Example 1. The prepared egg yolk active peptide product is marked as cAY1.

[0082] Comparative Example 2

[0083] Compared with Example 1, the neutral protease is omitted in the composite protease composition of this comparative example, and the mass ratio of alkaline protease to fermentation-type chymosin is 2:0.5. The remaining steps are the same as those of Example 1, and the prepared egg yolk active peptide product is labeled as cAY2.

[0084] Comparative Example 3

[0085] Compared with Example 1, the alkaline protease is omitted in the composite protease composition of this comparative example, and the mass ratio of neutral protease to fermentation-type chymosin is 1:0.5. The remaining steps are the same as those of Example 1, and the prepared egg yolk active peptide product is labeled as cAY3.

[0086] Comparative Example 4

[0087] Compared with Example 1, the composite protease composition of this comparative example omits fermentation-type chymosin, and the mass ratio of neutral protease to alkaline protease is 1:2. The remaining steps are the same as Example 1, and the prepared egg yolk active peptide product is labeled cAY4.

[0088] The present application conducts physical and chemical performance analysis on the egg yolk active peptide products prepared in the above-mentioned embodiments and comparative examples. The analysis results are shown in Table 1. The performance analysis methods are as follows:

[0089] Test experiment 1: Molecular weight analysis

[0090] In this embodiment, high performance liquid chromatography was used to detect the molecular weight distribution of egg yolk active peptide AY1. The chromatographic conditions were as follows: Waters 600 high performance liquid chromatograph (with 2487 UV detector and M32 workstation), TSKgelG 2000SW×L300mm×7.8mm, mobile phase V(acetonitrile):V(water):V(trifluoroacetic acid)=45:55:0.1, detection wavelength 220nm, flow rate 0.5mL / min, column temperature 30°C, and the molecular weight distribution diagram was shown in Table 1.

[0091] Table 1: Relative molecular weight distribution of egg yolk active peptide AY1

[0092]

[0093] Test experiment 2: Hydrolysis degree analysis

[0094] Determination of hydrolysis degree (OD): Method used: Double indicator formaldehyde titration method

[0095] Degree of hydrolysis (%) = (amount of amino nitrogen generated after hydrolysis / total nitrogen content in the sample) × 100%

[0096] Determination of total nitrogen content: Kjeldahl method

[0097] Test experiment 3: free amino acid analysis

[0098] Weigh 0.5 mL of sample into a hydrolysis tube, add 8 mL of 6 mol / L HCl solution, seal the tube with an alcohol burner under nitrogen protection, hydrolyze at 120 °C for 22 h, add 4.8 mL of 10 mol / L NaOH solution to neutralize, then make up to 25 mL with deionized water, filter, centrifuge at 15 000 r / min for 30 min, take the supernatant, filter with a 0.22 μm filter membrane, analyze with an amino acid analyzer, and calculate the proportion of free amino acids in the filtrate according to formula (1).

[0099] Free amino acid ratio / %= × 100 (1)

[0100] Table 2: Physical and chemical properties of egg yolk active peptide products

[0101]

[0102] According to Table 2, after adding 5 g / 100 g sodium sulfite to the egg yolk homogenized aqueous solution and performing ultrasonic shearing treatment, 32 g / 1000 g of a composite protease consisting of neutral protease, alkaline protease and fermentation-type chymosin in a mass ratio of 1:2:0.2 was added for composite enzymatic hydrolysis, which can make the product yield reach more than 70%, and can effectively regulate the hydrolysis degree and free amino acid proportion of egg yolk active peptides.

[0103] Test experiment 4: Mouse in vitro anti-shedding experiment

[0104] In order to illustrate the biological activity of the egg yolk active peptide prepared in the examples of the present application, an in vitro anti-hair loss experiment was carried out on mice using the egg yolk active peptide AY1 as a test sample.

[0105] Experimental method: 35 experimental mice were shaved on the back to remove hair (2cm × 2cm), and 7 mice were selected as the blank group and injected with an equal amount of saline subcutaneously. The rest were divided into a model group, a minoxidil group (positive control), and two experimental groups, and subcutaneously injected with DHT (dihydrotestosterone) 5mg / kg / day to create models; the blank group and the model group were treated with saline, the minoxidil group was sprayed with minoxidil spray 0.12ml / kg / day, the low-dose experimental group was applied with 3g / kg sample / day, and the high-dose experimental group was applied with 12g / kg / day sample; each group had 7 mice, and the drug was administered for 14 consecutive days, once a day. The hair growth of the mice was photographed on the 0th, 7th, 10th, and 14th days of drug administration, and the results were as follows: Figure 1 As shown; the mice were then killed on day 14, and the skin tissue was taken for HE staining to observe the hair follicles. The results were Figure 2 As shown. Among them, Figure 1 Images were taken of hair growth on the backs of experimental mice on different days; Figure 2 This is a HE staining image of the back skin tissue section of the experimental mouse.

[0106] according to Figure 1 to Figure 2 It can be seen that the comparison between the model group and the blank group showed that the AGA (androgenic alopecia) model was successfully established; the minoxidil group as a positive control had the best anti-hair loss effect; the experimental group significantly improved the hair growth rate and hair density compared with the blank group, and the hair growth and the number of hair follicles were positively correlated with the application dose, which shows that the egg yolk active peptide prepared in this application has a good effect of promoting hair growth.

[0107] Test experiment 5: HFDPC cell androgenic alopecia model experiment

[0108] AGA (androgenic alopecia) hair loss is caused by high levels of DHT (dihydrotestosterone) in the human body, which causes apoptosis of HFDPC (human hair follicle dermal papilla cells), resulting in abnormal hair cycle. Therefore, DHT-damaged HFDPC cells were used to establish an androgenic alopecia cell evaluation model. The evaluation model establishment method is as follows:

[0109] When the HFDPC cell growth density reached 80-90%, digestion and centrifugation were performed, and 100 μL of cell suspension was added to each well. The inoculation density was 5×10 3 / well. Cultured at 5% CO2 and 37℃ for 24h, the culture medium was aspirated, and washed twice with PBS. The cells were divided into a blank group (complete culture), a drug group (complete culture containing 120μmol / L DHT and different mass concentrations of AY1), a model group (complete culture containing 120μmol / L DHT) and a positive control group (complete culture containing 120μmol / L DHT and 5μmol / L minoxidil). Six replicate wells were set up in each group. Cultured at 5% CO2 and 37℃ for 48h, the supernatant was discarded, 100μL of 10% CCK-8 was added to each well, incubated at 37℃ in the dark for 4h, and the OD value was measured at 450 nm. The results were Figure 3 As shown, Figure 3 The relative viability of HFDPC cells varies with AY1 concentration.

[0110] according to Figure 3 It can be seen that compared with the blank group, the model group showed a significant inhibitory effect after 48 hours of administration (P<0.01); and compared with the model group, the positive control group could significantly improve cell viability (P<0.01). The egg yolk active peptide AY1 had no obvious effect on cell proliferation in the concentration range of 1-20μg / mL; but at a concentration of 40μg / mL, the relative cell viability was 70.5890% (P<0.05); at a concentration of 80μg / mL, the relative cell viability was 76.9443% (P<0.01), which could significantly improve the viability of HFDPC cells; at 160μg / mL, the relative cell viability was 70.0841% (P<0.05). According to the experimental results, the positive control group had a significant restorative effect on human hair follicle dermal papilla cells damaged by DHT; and the egg yolk active peptide AY1 also had a significant restorative effect on human hair follicle dermal papilla cells damaged by DHT in the concentration range of 40-160 μg / mL, especially at a concentration of 80 μg / mL, it showed the best protective effect on the cell proliferation of HFDPC, but the overall effect of the drug-treated group was lower than that of the positive control group.

[0111] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0112] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions recorded in the aforementioned embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. A method for preparing an egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells, characterized in that: The steps include: providing an egg yolk homogenate aqueous solution containing sulfite; Performing ultrasonic shearing treatment on the egg yolk homogenous aqueous solution to obtain a pre-treated substrate solution; Adding a composite protease at a rate of 32 g / 1000 g to the pretreated substrate solution for composite enzymatic hydrolysis to obtain an enzymatic hydrolysis product solution, wherein the composite protease is composed of a neutral protease, an alkaline protease and a fermentation-type chymosin in a mass ratio of 1:2:0.2; After the enzyme is inactivated, the enzymatic hydrolysis product solution is subjected to centrifugal separation and the lower layer is taken out and vacuum freeze-dried to obtain the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells.

2. The preparation method according to claim 1, characterized in that: The egg yolk homogenized aqueous solution is obtained by mixing the egg yolk stock solution and water in a mass ratio of 1:4 and then homogenizing; Furthermore, the mass percentage of the sulfite in the egg yolk homogenous aqueous solution is 5%.

3. The preparation method according to claim 1, characterized in that: When the ultrasonic shearing treatment is performed, the ultrasonic frequency is 20 kHz, the ultrasonic power is 420 W, and the ultrasonic time is 10 min.

4. The preparation method according to claim 1, characterized in that: When the composite enzymatic hydrolysis treatment is carried out, the temperature is maintained at 65-75° C., the pH value is maintained at 7-10, and the time is 1.5-3 hours.

5. An egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells prepared according to any one of the preparation methods of claims 1 to 4.

6. Use of the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells prepared by any one of the preparation methods of claims 1 to 4 or the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells according to claim 5 for stimulating hair growth in mammals in vitro.

7. The use according to claim 6, characterized in that: The effective concentration of the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells is 40-160 μg / mL.

8. The use according to claim 7, characterized in that: The effective concentration of the egg yolk active peptide for promoting the proliferation of hair follicle dermal papilla cells is 80 μg / mL.

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