Composite collagen peptide for promoting generation of collagen and elastin as well as preparation method and application of composite collagen peptide

Abstract

The invention belongs to the technical field of collagen peptides, and particularly relates to a composite collagen peptide for promoting generation of collagen and elastin as well as a preparation method and application of the composite collagen peptide. The preparation method comprises the following steps: selecting different raw materials to extract collagen crude extracts, proportioning the hydroxyproline content and the collagen crude extracts of different raw materials, performing enzymolysis by using compound protease, and separating and purifying enzymatic hydrolysate to obtain the compound collagen peptide. The composite collagen peptide provided by the invention can effectively promote the generation of collagen and elastin and promote skin fibroblasts to secrete type I collagen, type III collagen and elastin, and is safe and free of toxic and side effects. The composite collagen peptide provided by the invention has important significance in wide application in the fields of biomedicine, beauty and skin care, food and health care products and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of collagen peptides, and in particular relates to a composite collagen peptide that promotes the production of collagen and elastin, and a preparation method and application thereof. Background Art

[0002] Skin is the largest organ in the human body. It is located on the outermost part of the body. Collagen and elastin in the skin are two extremely important structural proteins. Together, they constitute the extracellular matrix (ECM) of the skin, providing the skin with necessary structural support and functional properties. Human skin contains many different types of collagen, including type I and type III collagen, which have different functions and effects. Type I and type III collagen have the highest content. Type I collagen accounts for about 80% of the dermis of the skin, making the skin plump and full. Type III collagen, which belongs to the fibril-forming collagen, accounts for 10% of the dermis collagen. It combines with type I collagen to form collagen fiber bundles, which are mainly distributed in the papillary and reticular areas of the dermis. The main function of collagen is to provide structural support for the skin and maintain the firmness and elasticity of the skin. Elastin is another important skin structural protein. Together with collagen, it maintains the elasticity and flexibility of the skin, just like the steel bars in a house. Elastin is interwoven into a mesh structure in the dermis, allowing the skin to return to its original shape after being stretched by external forces. Compared to collagen, elastin is slower to renew, but more stretchable. Collagen and elastin interact in the skin, forming a complex network that supports the skin's structure. Collagen provides the skin's strength and toughness, while elastin gives it elasticity. A balance between the two is essential to keep the skin healthy and youthful.

[0003] As we age, the synthesis of collagen and elastin gradually decreases, and UV exposure and other environmental factors accelerate the degradation of collagen and elastin, leading to wrinkle formation, decreased elasticity and skin sagging during skin aging.

[0004] Patent CN118388666A discloses a polypeptide that can simultaneously promote the regeneration of skin collagen and elastin, but the polypeptide is prepared by genetically engineered bacteria and its application in food is currently restricted by laws and regulations. Patent CN116617122 A discloses a composition that can promote the synthesis and regeneration of type I, type III, type V, type IV, type VI, type XII, type VII, type XVII and type XVIII collagen in skin cells, but it does not promote the synthesis of elastin, so its effect has certain limitations.

[0005] Therefore, there is an urgent need to develop an active ingredient that can synergistically enhance the synthesis of collagen and elastin, thereby adjusting the skin condition, especially an active ingredient that can simultaneously promote the synthesis of type I collagen, type III collagen and elastin, and has high safety and good absorption. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a composite collagen peptide that promotes the production of collagen and elastin and a preparation method thereof. The prepared composite collagen peptide can effectively promote the production of collagen and elastin, can significantly promote the secretion of type I collagen, type III collagen and elastin by skin fibroblasts, is safe and has no toxic side effects, and can be widely used in various fields such as ordinary food, health food and cosmetics.

[0007] In one aspect, the present invention provides a method for preparing a composite collagen peptide, comprising the following steps:

[0008] S1. Defatting different raw materials respectively, and extracting with boiling water to obtain crude collagen extracts of different raw materials;

[0009] S2. According to the hydroxyproline content, the crude collagen extracts of the above different raw materials are proportioned to obtain a composite crude collagen extract;

[0010] S3, adding a composite protease to the composite collagen crude extract for enzymolysis to obtain an enzymolysis solution;

[0011] S4, inoculating Bacillus into the above enzymatic hydrolysate, fermenting, and obtaining collagen peptide fermentation liquid;

[0012] S5, separating and purifying the aforementioned collagen peptide fermentation broth to obtain a collagen peptide solution;

[0013] S6, spray-drying the above collagen peptide solution to obtain collagen peptide powder, i.e. composite collagen peptide;

[0014] The hydroxyproline content in step S2 is 10wt%-12wt%.

[0015] Specifically, the raw materials in step S1 include but are not limited to: animal skins, scales or bones.

[0016] More specifically, the animals in step S1 include but are not limited to one or more of fish, pigs, cattle, sheep or poultry.

[0017] Preferably, the raw materials in step S1 include cow hide and cow bones.

[0018] Specifically, the leaching solution in step S1 is a NaOH solution.

[0019] Further specifically, the mass fraction of the NaOH solution is 0.1wt%-2.5wt%;

[0020] The mass ratio of the raw material to the NaOH solution may be 1:5-1:20;

[0021] The extraction time is 12h-24h.

[0022] Specifically, the composite protease in step S3 includes alkaline protease, pepsin and bromelain.

[0023] Specifically, the mass ratio of alkaline protease, pepsin and bromelain can be 2.5-4:0.8-1.5:1.

[0024] In some embodiments, the mass ratio of alkaline protease, pepsin and bromelain can be 2.5:0.8:1.

[0025] In other embodiments, the mass ratio of alkaline protease, pepsin and bromelain can be 4:1.5:1.

[0026] Specifically, the enzyme activity of the alkaline protease is 150,000-205,000 U / g, the enzyme activity of the pepsin is 1000-1500 U / g, and the enzyme activity of the bromelain is 95,000-105,000 U / g, or they are converted into the enzyme activities for use.

[0027] Preferably, the enzyme activity of the alkaline protease is 200,000 U / g, the enzyme activity of the pepsin is 1200 U / g, and the enzyme activity of the bromelain is 100,000 U / g, or they are converted into the enzyme activities for use.

[0028] Specifically, the dosage of the composite protease is 0.5wt%-2.5wt%.

[0029] Specifically, the enzymatic hydrolysis conditions of the composite protease are: 55°C-75°C, 2h-8h, and pH 7.0-9.0.

[0030] Specifically, the Bacillus in step S4 includes one or more of Bacillus licheniformis, Bacillus gelatinus or Bacillus siamese.

[0031] Preferably, the Bacillus in step S4 is Bacillus siamese; the inoculum amount of the Bacillus can be 10 8 cfu / g-10 9 cfu / g.

[0032] Specifically, the fermentation conditions in step S4 are: 35°C-42°C, 48h-96h.

[0033] Specifically, the separation and purification in step S4 includes one or more of ultrafiltration or nanofiltration.

[0034] More specifically, the ultrafiltration membrane is any one of polysulfone and polyamide rolled membranes; the nanofiltration membrane is any one of polyamide, sulfonated polyethersulfone and sulfonated polysulfone hollow fiber membranes.

[0035] More specifically, the molecular weight cutoff of the ultrafiltration membrane used may be 1KDa-10kDa; the molecular weight cutoff of the nanofiltration membrane may be 100Da-2000Da.

[0036] Specifically, the inlet air temperature of the spray drying in step S6 is set to 160°C-220°C, and the outlet air temperature is set to 90°C-120°C.

[0037] Specifically, the average molecular weight of the collagen peptide powder in step S6 is 500Da-3000Da, wherein the peptides of 500Da-1000Da account for more than 50wt%.

[0038] More specifically, the amino acid composition of 500Da-3000Da includes: 20wt%-27wt% of glycine, 10wt%-15wt% of proline, 5wt%-12wt% of glutamic acid, 5wt%-12wt% of arginine, and 2wt%-7wt% of aspartic acid.

[0039] Preferably, the amino acid composition of 500Da-3000Da includes: 24wt%-27wt% of glycine, 11wt%-13wt% of proline, 8wt%-10wt% of glutamic acid, 8wt%-10wt% of arginine, and 5wt%-6wt% of aspartic acid.

[0040] In some embodiments, the amino acid composition of 500Da-3000Da includes: 27wt% of glycine, 11wt% of proline, 8wt% of glutamic acid, 10wt% of arginine, and 6wt% of aspartic acid.

[0041] In other embodiments, the amino acid composition of 500Da-3000Da includes: 24wt% of glycine, 13wt% of proline, 10wt% of glutamic acid, 8wt% of arginine, and 5wt% of aspartic acid.

[0042] In yet another aspect, the present invention provides a composite collagen peptide, wherein the composite collagen peptide is obtained by the aforementioned preparation method.

[0043] In another aspect, the present invention provides the use of the aforementioned composite collagen peptide in the preparation of medicines, foods or cosmetics for promoting the production of collagen and elastin.

[0044] In yet another aspect, the present invention provides a food, comprising the aforementioned composite collagen peptide.

[0045] Specifically, the food includes but is not limited to solid beverages and liquid beverages.

[0046] Specifically, the food also includes excipients that are acceptable in food science.

[0047] More specifically, the food-scientifically acceptable auxiliary material can be selected from one or more of desalted whey powder, skimmed milk powder, refined vegetable oil, α-lactalbumin, maltodextrin, lactose, fructooligosaccharides, nucleotides, whey protein peptides, β-carotene, choline, L-carnitine, multivitamins, edible minerals and galacto-oligosaccharides.

[0048] Preferably, the complex vitamin can be selected from a combination of one or more of vitamin A (retinol), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid), vitamin B12 (cyanocobalamin), vitamin C (ascorbic acid), vitamin D (lamisterol), vitamin D2 (calciferol), vitamin D3 (dihyrotachysterol), vitamin D4 (7-dehydrositosterol), vitamin E (tocopherol), and vitamin K (naphthoquinone).

[0049] Preferably, the edible minerals may be selected from one or more of calcium chloride, magnesium, phosphorus, potassium, sodium, and sulfur.

[0050] In yet another aspect, the present invention provides a medicine comprising the aforementioned composite collagen peptide.

[0051] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0052] More specifically, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0053] Preferably, the pharmaceutically acceptable excipient is at least one selected from lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate and mineral oil.

[0054] In yet another aspect, the present invention provides a cosmetic, characterized in that the cosmetic comprises the aforementioned composite collagen peptide.

[0055] The technical effects achieved by the present invention are:

[0056] (1) In cell experiments, it can effectively and significantly promote the secretion of type I collagen, type III collagen and elastin by skin fibroblasts.

[0057] (2) The collagen peptides provided in the present application have an average molecular weight of 500Da-3000Da. According to molecular weight distribution detection, peptides of 500Da-1000Da account for more than 50%, and are rich in glycine, proline, glutamic acid, arginine, and aspartic acid (wherein glycine accounts for 20wt%-27wt%, proline accounts for 10wt%-15wt%, glutamic acid accounts for 5wt%-12wt%, arginine accounts for 5wt%-12wt%, and aspartic acid accounts for 2wt%-7wt%).

[0058] (3) Third, the collagen peptide provided in the present application is safe and has no toxic side effects. It is not only easy to digest and absorb orally, but also can be absorbed through the skin. It can be widely used in various fields such as ordinary food, health food and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The effect of different collagen peptides on the expression of type I collagen in human fibroblasts.

[0060] Figure 2 The effect of different collagen peptides on the expression of type III collagen in human fibroblasts.

[0061] Figure 3 The effect of different collagen peptides on the expression of elastin in human fibroblasts.

[0062] Figure 4 The effect of different collagen peptides on the secretion of type I collagen in human fibroblasts.

[0063] Figure 5 The effect of different collagen peptides on the secretion of type III collagen in human fibroblasts.

[0064] Figure 6 Effects of different collagen peptides on elastin secretion in human fibroblasts.

[0065] Figure 7 It is the cumulative permeation per unit area of ​​different collagen peptides. DETAILED DESCRIPTION

[0066] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0067] The calfskin described in the present invention refers to the hide of cattle that is less than 1 year old, regardless of gender.

[0068] The ordinary cowhide described in the present invention refers to the hide of adult cows over 2 years old.

[0069] In the present invention, the enzyme activity of alkaline protease is 200,000 U / g, the enzyme activity of pepsin is 1200 U / g, and the enzyme activity of bromelain is 100,000 U / g, or they are converted into the enzyme activities for use.

[0070] Example 1

[0071] A method for preparing a composite collagen peptide for promoting the production of collagen and elastin, the specific steps comprising:

[0072] S1. Soak 1 kg of collagen raw materials from different sources (dairy cowhide, ordinary cowhide, cow bone) in 20 kg of 2.5 wt% NaOH solution for 12 h, wash with water until neutral, repeat 3 times, take out the material and add it back into 10 kg of water, and extract at 100 ° C for 6 hours to obtain a crude collagen extract.

[0073] S2. Detect the collagen content (expressed as hydroxyproline content) and amino acid composition in the crude collagen extracts, and according to the hydroxyproline content, mix the three crude collagen extracts so that the hydroxyproline content is between 10wt%.

[0074] S3. Alkaline protease, pepsin and bromelain are mixed in a mass ratio of 2.5:0.8:1 to prepare a composite protease.

[0075] S4. Add water to the compounded collagen crude extract, adjust the pH value of the compounded collagen crude extract solution to about 8.0, add 2.5wt% of the compound protease by weight of the compounded collagen crude extract; perform enzymolysis at 60°C for 5 hours, inactivate the enzyme, and obtain an enzymolysis solution.

[0076] S5. Adjust the pH value of the enzymatic solution to about 6.0. 9 The mixture was inoculated with Bacillus siamese at an inoculum amount of 1.0 cfu / g, and then fermented at 37°C for 72 hours to obtain a collagen peptide fermentation liquid.

[0077] S6. After solid-liquid separation of the fermentation broth, the filtrate is collected and filtered through an ultrafiltration membrane and nanofiltration (molecular weight cutoff 500Da-3000Da) to obtain a collagen peptide solution, which is then spray-dried (the inlet air temperature is set to 160°C and the outlet air temperature is set to 90°C) to obtain a collagen peptide powder that promotes the production of collagen and elastin.

[0078] The molecular weight of small molecule collagen peptides was tested according to the method described in GB 31645-2018 National Food Safety Standard Collagen Peptides, and its average molecular weight was 1164Da. According to the molecular weight distribution test, peptides of 500Da-1000Da accounted for 53%, of which glycine accounted for 27wt%, proline accounted for 11wt%, glutamic acid accounted for 8wt%, arginine accounted for 10wt%, and aspartic acid accounted for 6wt%.

[0079] Example 2

[0080] A method for preparing a composite collagen peptide for promoting the production of collagen and elastin, the specific steps comprising:

[0081] S1. Soak 1 kg of collagen raw materials from different sources (dairy cowhide, ordinary cowhide, cow bone) in 15 kg of 0.5% NaOH solution for 12 hours, wash with water until neutral, repeat 3 times, take out the material and add it back into 10 kg of water, and extract at 100°C for 6 hours to obtain a crude collagen extract.

[0082] S2. Detect the collagen content (expressed as hydroxyproline content) and amino acid composition in the crude collagen extracts, and according to the hydroxyproline content, mix the three crude collagen extracts so that the hydroxyproline content is between 12wt%.

[0083] S3. Alkaline protease, pepsin and bromelain are mixed in a mass ratio of 4:1.5:1 to prepare a composite protease.

[0084] S4. Add water to the composite collagen crude extract, adjust the pH value of the composite collagen crude extract solution to about 9, add 0.5wt% of the composite protease by weight of the composite collagen crude extract; perform enzymolysis at a temperature of 70°C for 8 hours, inactivate the enzyme, and obtain an enzymolysis solution.

[0085] S5. Adjust the pH value of the enzymatic solution to about 6.0. 8The mixture was inoculated with Bacillus siamese at an inoculum amount of 1.0 cfu / g, and then fermented at 40°C for 96 hours to obtain a collagen peptide fermentation liquid.

[0086] S6. After solid-liquid separation of the fermentation broth, the filtrate is collected and filtered through an ultrafiltration membrane and nanofiltration (molecular weight cutoff 500Da-3000Da) to obtain a collagen peptide solution, which is then spray-dried (the inlet air temperature is set to 220°C and the outlet air temperature is set to 120°C) to obtain a collagen peptide powder that promotes the production of collagen and elastin.

[0087] The molecular weight of small molecule collagen peptides was tested according to the method described in GB 31645-2018 National Food Safety Standard Collagen Peptides, and its average molecular weight was 1164Da. According to the molecular weight distribution test, 500Da-1000Da peptides accounted for 58%, of which glycine accounted for 24wt%, proline accounted for 13wt%, glutamic acid accounted for 10wt%, arginine accounted for 8wt%, and aspartic acid accounted for 5wt%.

[0088] Comparative Example 1

[0089] A method for preparing a composite collagen peptide that promotes the production of collagen and elastin, wherein the steps are different from those of Example 1 in that the raw material of step S1 is only dairy cow hide, and there is no need to compound the raw materials in step S2, and the remaining steps are the same as those of Example 1.

[0090] Comparative Example 2

[0091] A method for preparing a composite collagen peptide that promotes the production of collagen and elastin, wherein the steps are different from those of Example 1 in that Bacillus siamese is not inoculated for fermentation in step S4. The remaining steps are the same as those of Example 1.

[0092] Effect Example 1 Real-time PCR detection of the effect of collagen peptides on the expression of type I collagen, type III collagen and elastin-related genes in human fibroblasts

[0093] After the cells have spread all over the plate, collect the cells into a 10 mL centrifuge tube, centrifuge at 1200 rpm for 3 min, remove the supernatant, add new cell culture medium, pipette evenly, and count using a hemocytometer. In each well of a 12-well plate, spread 2.5 × 10 5Cells were cultured overnight. Then, cell culture fluid without any collagen peptides was added (blank control), cell culture fluid with collagen peptides of Comparative Example 1 (Comparative Example 1), cell culture fluid with collagen peptides of Comparative Example 2 (Comparative Example 2), cell culture fluid with commercially available collagen peptides (commercially available), cell culture fluid of Example 1 (Example 1), and cell culture fluid of Example 2 (Example 2) were added to treat the cells for 4 hours. Cell RNA was extracted according to the steps in the instruction manual, and cDNA was synthesized by reverse transcription. The reagents were configured according to the following reaction system for RT-qPCR experiments.

[0094] Table 1. Primer sequences for Real-time PCR detection

[0095]

[0096] Table 2. Composition of PCR system for real-time PCR detection

[0097]

[0098]

[0099] Table 3. Real-time PCR detection PCR reaction program

[0100]

[0101] The results are as follows Figure 1-Figure 3 As shown, adding different collagen peptides to the cell culture medium can significantly increase the expression levels of type I collagen, type III collagen and elastin in human fibroblasts, among which the increase values ​​in Example 1 and Example 2 are higher.

[0102] Effect Example 2 Western Blot Detection of the Effect of Collagen Peptide on the Secretion of Type I Collagen, Type III Collagen and Elastin in Human Fibroblasts

[0103] The detection steps are as follows:

[0104] (1) Sample processing and collection: After the cells are fully spread on the plate, collect the cells into a 10 mL centrifuge tube, centrifuge at 1200 rpm for 3 min, remove the supernatant, add new cell culture medium, pipette evenly, and count using a hemocytometer. In each well of a 12-well plate, spread 2.5 × 10 5Cells were cultured overnight. After that, cell culture fluid without any collagen peptide was added (blank control), cell culture fluid with collagen peptide of comparative example 1 (comparative example 1), cell culture fluid with collagen peptide of comparative example 2 (comparative example 2), cell culture fluid with commercially available collagen peptide (commercially available), cell culture fluid of example 1 (example 1) and cell culture fluid of example 2 (example 2) were added to treat the cells for 4 hours, so that the final concentration of collagen 1 was 200 μg / mL, the final concentration of collagen 2 was 800 μg / mL, the final concentration of type III collagen 1 was 800 μg / mL, the final concentration of type III collagen 2 was 100 μg / mL, and the final concentration of type III collagen 3 was 100 μg / mL, and 3 parallels were set for each concentration. After the cell treatment was completed, the cell lysis solution was added to fully lyse the cells, and then the protein loading buffer was added and mixed, and then collected into a 1.5 mL centrifuge tube, heated at 100 ° C for 10 min, and stored at -20 ° C for use.

[0105] (2) Preparation of SDS-PAGE electrophoresis gel: Prepare 10% separation gel according to the recipe, seal and flatten the separation gel with 75% ethanol, let it stand for 20 minutes until the gel solidifies, pour out the ethanol solution, add the concentrated gel and insert the comb, let it stand for more than 20 minutes until it solidifies before conducting the electrophoresis experiment.

[0106] (3) SDS-PAGE protein electrophoresis: clamp the electrophoresis gel and pour in the prepared electrophoresis buffer, unplug the comb, add the protein maker and protein sample in order, and perform electrophoresis at 100V constant voltage for 1.5h. After the electrophoresis is completed, cut a PVDF membrane of appropriate size and soak it in anhydrous methanol for 3min. Place it in the transfer tank in the order of sponge → filter paper → gel → membrane → filter paper → sponge, fill it with the prepared transfer buffer, and transfer it at 100V at 4℃ for 1.5h. After the transfer, use 5% skim milk prepared in TBST to block the membrane at room temperature for 2h. Dilute the corresponding primary antibody according to the ratio and incubate it on a shaker at 4℃ overnight. The primary antibody (Abcam (Shanghai) Trading Co., Ltd.) was recovered the next day, and the membrane was washed 3 times with TBST for 10min each time. The secondary antibody (Abcam (Shanghai) Trading Co., Ltd.) was diluted 1:2000 and incubated on a shaker at room temperature for 1.5h. Discard the secondary antibody, wash the membrane three times with TBST, 10 min each time, add ECL chemiluminescent colorimetric solution, and use a chemiluminescent gel imager to image.

[0107] The results are as follows Figure 4-Figure 6 As shown, adding different collagen peptides to the cell culture medium can significantly increase the secretion of type I collagen, type III collagen and elastin of human fibroblasts, among which the increased values ​​of Example 1 and Example 2 are higher.

[0108] Effect Example 3 Transdermal Absorption Experiment

[0109] The transdermal absorption experiment was conducted using a TP-6 ​​transdermal diffusion instrument (manufacturer: Tianjin Tianhe Analytical Instrument Co., Ltd.). The skin-specific membrane (manufacturer: Merck Millipore, USA) is directly fixed between the sample pool and the receiving pool. The sample pool contains a 10% collagen peptide saline solution, and saline is used as a blank control. Fill the receiving pool with saline to a capacity of 17mL, maintain constant stirring and constant temperature, water bath temperature (37℃±0.5℃), and rotation speed of 300rpm. Take 1mL of sample at 2h, 4h, 6h, 8h and 24h respectively, and add an equal amount of saline at the same time, and then take a certain amount of test solution from the receiving solution and place it in a volumetric flask. Determine the content of Hyp in the extracted liquid, and calculate the cumulative permeation per unit area (Q n ) and transdermal rate constant (J):

[0110]

[0111] In the formula, C n is the Hyp concentration measured at the nth sampling point, in μg / mL; C i is the Hyp concentration measured at the ith sampling point, in μg / mL; 10 is the conversion coefficient of hyp and collagen peptide; V is the capacity of the receiving pool, which is 17 mL; V0 is the volume taken at each time point, which is 1 mL; A is the penetration area, which is 1.54 cm2.

[0112]

[0113] Cumulative penetration Q n Linear regression was performed on time t, and the slope of the equation was the permeation rate constant (μg / cm 2 / h).

[0114] The results are as follows Figure 7 As shown, the cumulative permeation per unit area of ​​the five collagen peptides tested increased with time, among which the cumulative permeation per unit area of ​​Example 1 and Example 2 was high, and the transdermal absorption capacity of Example 1 and Example 2 was better than that of the other three collagen peptides.

Claims

1. A method for preparing a composite collagen peptide, characterized in that: The following steps are involved: S1. Defatting different raw materials respectively, and extracting with boiling water to obtain crude collagen extracts of different raw materials; S2. According to the hydroxyproline content, the crude collagen extracts of the above different raw materials are proportioned to obtain a composite crude collagen extract; S3, adding a composite protease to the composite collagen crude extract for enzymolysis to obtain an enzymolysis solution; S4, inoculating Bacillus into the above enzymatic hydrolysate, fermenting, and obtaining collagen peptide fermentation liquid; S5, separating and purifying the aforementioned collagen peptide fermentation broth to obtain a collagen peptide solution; S6, spray-drying the above collagen peptide solution to obtain collagen peptide powder, i.e. composite collagen peptide; The hydroxyproline content in step S2 is 10%-12wt%.

2. The preparation method according to claim 1, characterized in that: The raw materials in step S1 include animal skins, scales or bones.

3. The preparation method according to claim 2, characterized in that: The animals in step S1 include one or more of fish, pigs, cattle, sheep or poultry.

4. The preparation method according to claim 1, characterized in that: The composite protease in step S3 comprises alkaline protease, pepsin and bromelain.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the alkaline protease, pepsin and bromelain is 2.5-4:0.8-1.5:

1.

6. The preparation method according to claim 5, characterized in that: The enzyme activity of the alkaline protease is 150,000-205,000 U / g, the enzyme activity of the pepsin is 1000-1500 U / g, and the enzyme activity of the bromelain is 95,000-105,000 U / g, or they can be converted into the enzyme activities for use.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The dosage of the composite protease is 0.5wt%-2.5wt%.

8. The preparation method according to claim 7, characterized in that: The enzymatic hydrolysis conditions of the composite protease are: 55° C.-75° C., 2 h-8 h, and pH value 7.0-9.

0.

9. The preparation method according to claim 1, characterized in that: The Bacillus in step S4 includes one or more of Bacillus licheniformis, Bacillus gelatinus or Bacillus siamese.

10. The preparation method according to claim 9, characterized in that: The Bacillus in step S4 is Siamese Bacillus; the inoculum amount of the Bacillus is 10 8 cfu / g-10 9 cfu / g.

11. The preparation method according to claim 1, characterized in that: The fermentation conditions in step S4 are: 35° C.-42° C., 48 h-96 h.

12. The preparation method according to claim 1, characterized in that: The average molecular weight of the collagen peptide powder in step S6 is 500Da-3000Da, wherein the peptides of 500Da-1000Da account for more than 50wt%.

13. The preparation method according to claim 12, characterized in that: The amino acid composition of the 500Da-3000Da peptide includes: 20wt%-27wt% of glycine, 10wt%-15wt% of proline, 5wt%-12wt% of glutamic acid, 5wt%-12wt% of arginine, and 2wt%-7wt% of aspartic acid.

14. A composite collagen peptide, characterized in that: The composite collagen peptide is obtained by the preparation method according to any one of claims 1 to 13.

15. Use of the composite collagen peptide according to claim 14 in the preparation of medicines, foods or cosmetics for promoting the production of collagen and elastin.

16. A food, characterized in that The food comprises the composite collagen peptide according to claim 14.

17. A drug, characterized in that The medicine comprises the composite collagen peptide according to claim 14.

18. A cosmetic, characterized in that: The cosmetic comprises the composite collagen peptide according to claim 14.

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