A collagen tripeptide composition and purification method thereof

Abstract

The present invention provides a process for extracting collagen tripeptide (CTP) from collagenase hydrolysate, comprising the following steps: performing two enzymatic hydrolysis on the pretreated collagen raw material, filtering, and adopting specific magnetic microsphere separation and molecular imprinting polymer microsphere medium solid phase extraction purification. The present invention can obtain a high-content (up to 80%) collagen tripeptide composition product (Gly-X-Y, including Gly-Pro-Hyp). The present invention has the advantages of high separation efficiency, low cost, and suitability for industrial-scale production. Studies have shown that the compound use of collagen tripeptide and elastin peptide in a ratio of 16:1 to 24:1 can significantly increase the content of elastin and collagen in the skin, and significantly increase the content of hyaluronic acid and hydroxyproline, while reducing the content of MMP3 and inhibiting skin inflammatory factors.

Description

Technical Field

[0001] The invention belongs to a method for extracting, separating and purifying high-purity collagen tripeptides, and particularly relates to a purification method for extracting and separating high-purity collagen tripeptides from fish scale or fish skin enzymatic hydrolysate. Background Art

[0002] Collagen is a fibrous protein composed of various amino acids, including glycine, proline, and hydroxyproline. It has a high molecular weight of approximately 300 kDa (kilodaltons). This is higher than most proteins (which typically have molecular weights below 100 kDa), making it difficult for humans to digest and absorb. It typically needs to be degraded into low-molecular-weight collagen peptides with a molecular weight below 5000 for use. Low-molecular-weight collagen peptides, also known as collagen, can be degraded in the body and absorbed as amino acids.

[0003] Since artificial synthesis of collagen peptides is expensive and difficult to operate, low molecular weight collagen peptides are currently mainly obtained by protein hydrolysis. Small molecule peptides, especially small molecular weight peptides formed by dehydration condensation of 3 or more amino acids, are easy to absorb. Collagen peptides usually have a unique repeating sequence structure rich in glycine-proline-hydroxyproline (or glycine-xy). The small peptide formed by the basic unit of this repeating sequence structure is called collagen tripeptide.

[0004] (CTP), so the molecular weight of collagen tripeptide is usually around 200-300 Da, and its derivatives (e.g., incomplete hydrolysis) do not exceed 1000 Da. Due to its small molecular weight, it is easily absorbed through the skin. When the molecular weight exceeds 800 or 1000 Da due to the connection of multiple amino acids, it cannot be absorbed through the skin quickly due to the increased molecular weight. Therefore, collagen tripeptide (glycine-xy) has a wide range of demand and value in the fields of medicine, beauty and skin care products, health products, food, etc.

[0005] Currently, the raw materials for collagen extraction are mainly animal skins, such as cowhide, pigskin, fish skin and other tissues with high fat content. Collagen tripeptide compositions are obtained through a series of treatments including enzymatic hydrolysis. However, the content of tripeptides in the resulting product is very low (in addition, the existing technology is not very effective in removing grease, resulting in residual grease, which in turn affects the quality of the collagen tripeptides). Currently, a variety of composite proteases have been developed for the extraction and preparation of collagen peptides. The most widely used proteases include alkaline protease, neutral protease, papain, trypsin, pepsin, and flavor protease.

[0006] However, due to the nature of enzymatic hydrolysis, high yields of collagen tripeptide molecules cannot be achieved. Furthermore, the isolation and purification of collagen tripeptides is difficult and costly, resulting in a low final extraction rate and low purity. The content of collagen tripeptide derivatives in peptide products obtained using existing technologies is typically below 20%, making further purification impossible to achieve a higher purity, limiting their application.

[0007] Therefore, there is a need for an efficient separation and purification method for preparing high-content collagen tripeptide (glycine-xy).

[0008] CN 107532157 A discloses a method for preparing collagen tripeptide. The steps are conventional, but it uses a specific collagenase, the extraction process is cumbersome, and compared with common enzymes on the market, the operation is inconvenient.

[0009] CN 113789360 A relates to a method for extracting collagen tripeptides and the prepared collagen tripeptides. The method employs an enzymatic method to extract collagen peptides from fish skin by using a mixture of alkaline protease and papain in a 1:3 ratio. Simultaneously, the fish skin is treated with 600W ultrasonic waves for 45 minutes. This method increases the extraction rate of collagen peptides from the fish skin by 15% to 25%. However, this method does not yield high levels of collagen peptides.

[0010] CN 108929381 A provides a collagen tripeptide for emergency skin repair and a preparation method thereof. The collagen tripeptide is prepared using gelatin from mussels as a raw material through a double-enzyme hydrolysis method using enzymes selected from bromelain, papain, ficin, and gingerase. However, the obtained tripeptide is not highly pure.

[0011] The above-mentioned prior art basically improves the small molecule peptide content of collagen hydrolysate by improving the collagen hydrolysis process or the combination of enzymes used while continuing to use the existing preparation process, and by using new enzymes or a combination of enzymes.

[0012] Molecularly imprinted polymers (MIPs) are polymers that have specific adsorption capabilities for target molecules. Currently, MIP separations are widely used in solid-phase extraction, separation, and purification. However, there is currently no evidence that MIPs can enhance selective adsorption for peptide separations.

[0013] Given that the above-mentioned prior art for the purification of collagen tripeptides does not involve a description of large-scale separation and batch processing from an enzymatic hydrolysate to obtain a high-purity product (the collagen tripeptide content in most small-molecule polypeptide products is not high, generally not exceeding 20%). Therefore, when faced with an enzymatic hydrolysate with complex components, the prior art is difficult to directly purify and separate high-purity collagen tripeptides (glycine-xy) therefrom by ion exchange, silica gel or membrane filtration. Therefore, a collagen tripeptide extraction and separation technology based on collagen enzymatic hydrolysate raw materials, with low development costs, suitable for industrial large-scale separation and purification, and with high product purity, has important market value. Summary of the Invention

[0014] To address the problems of low separation efficiency and low product purity in the prior art of extracting collagen tripeptides (CTP) from collagenase hydrolysates, the present invention provides a collagen tripeptide separation and purification process based on a combination of magnetic microsphere polypeptide separation and solid-phase specific adsorption extraction to obtain a high-content collagen tripeptide product (Gly-XY, including Gly-Pro-Hyp). This method has the advantages of high separation efficiency, easy access to raw materials, low cost, and suitability for industrial-scale production.

[0015] Another object of the present invention is to provide a high-content or high-purity collagen tripeptide composition product, which has a content of small molecule collagen peptides (molecular weight not greater than 1KDa) of not less than 95%; wherein the content of collagen tripeptide (Gly-XY, X is Pro or Hyp, Y is other amino acids) in the obtained composition is not less than 80%.

[0016] The main steps adopted in the method of the present invention are:

[0017] The pre-treated collagen raw material is enzymatically hydrolyzed twice, and then the macromolecular impurities that are not fully hydrolyzed are removed through a nanofiltration membrane. Then, a specific magnetic microsphere separation and molecularly imprinted polymer microsphere medium solid phase extraction are combined to purify and separate. Specifically, it includes:

[0018] 1) crushing and heating the collagen raw material, and then subjecting it to acidic enzymatic hydrolysis and alkaline enzymatic hydrolysis in sequence; the enzymatic hydrolysis includes at least one collagenase;

[0019] 2) After the enzymatic hydrolyzate is subjected to nanofiltration or ultrafiltration, nonspecific adsorption separation is performed using at least one magnetic microsphere filler medium selected from surface-modified or unmodified polymethacrylate-based magnetic microspheres or PVA (polyvinyl alcohol)-based magnetic microspheres; preferably, a composite filler of the two microspheres is used; further preferably, silica gel or mesoporous silica is also used for pre-separation treatment;

[0020] 3) Gradient solid-phase extraction purification using molecular imprinting: The small molecule collagen peptides separated by nonspecific adsorption are loaded onto molecularly imprinted polymer separation columns prepared with different template molecules for specific separation and purification to obtain a composition high in collagen tripeptide content. The different template molecules are selected from the group consisting of a tripeptide template molecule with a Gly terminal group, a dipeptide template molecule with a Gly terminal group, and characteristic amino acids of a collagen tripeptide.

[0021] The characteristic amino acids of the collagen tripeptide are selected from at least one of glycine and hydroxyproline, preferably hydroxyproline or both.

[0022] The molecularly imprinted polymer is prepared by using the above template molecule in the presence of a functional monomer and a cross-linking agent using a thermal polymerization technique. The preparation method of molecularly imprinted polymer is well known in the art.

[0023] The collagen protein raw material described herein is preferably selected from fish scales, defatted fish skin, or a mixture of the two. It can also be selected from scaled fish skin. Considering the cost of the raw material and the operating costs of the pretreatment process, fish scales, especially freshwater fish scales, are more preferred. When a mixture of the two is used, the defatted fish skin content does not exceed 50% of the total weight of the raw material (based on the dry weight of the raw material).

[0024] In addition, methods for defatting fish skin are also known in the prior art.

[0025] Specifically, the technical solutions of the present invention are as follows.

[0026] In a first aspect, the present invention provides a method for separating and purifying high-purity collagen tripeptide, comprising the following steps S1-S5:

[0027] Step S1: Pretreatment of collagen raw materials

[0028] The collagen source material is selected from fish scales, defatted fish skin (excluding scales), or a mixture of the two (which may also include fish skin containing scales). Fish scales, such as freshwater fish scales, are preferred, as they are low-cost and low in fat. When a mixture of the two is used, the defatted fish skin content preferably does not exceed 30% of the total weight of the raw materials (based on dry weight).

[0029] When fish scales are used as raw materials, they can be directly pulverized with water to prepare pulp, or they can be washed, dried, pulverized, and sieved (preferably not less than 40 mesh) (to facilitate batch storage of raw materials during large-scale preparation), and then pretreated according to the following steps:

[0030] The crushed fish scale powder is fully soaked in water at a temperature ranging from room temperature to 50-60°C, and then removed and drained; the scales are soaked in a 0.1-0.3M (preferably 0.1-0.2M) HCl solution equivalent to 5-15 times the weight of the scales at room temperature for 3-5 hours, and then rinsed with clean water; the cleaned scales are placed in a heating kettle with stirring, 5-10 times the weight of deionized water is added, and the scales are heated at a temperature of 95-100°C with stirring for 6-8 hours to obtain a fish scale glue solution.

[0031] The obtained fish scale gelatin solution is filtered while hot to remove solid impurities; the filtrate is cooled to room temperature for later use to obtain a crude collagen gelatin solution.

[0032] When defatted fish skin is used as raw material, pretreatment is performed according to the following steps:

[0033] The fish skin after 0.1-0.2M NaOH solution soaking treatment and soaking the degreasing with an organic solvent is pulverized and sieved (preferably not less than 20 orders) after the dehydration and drying;

[0034] Exemplarily, the defatting step is as follows: soaking the fish skin in a 0.1-0.2M NaOH solution (5-6 times the weight of the fish skin raw material) at a soaking temperature of 25°C for 5-10 hours to remove foreign proteins and fat, washing it with water, then soaking it in a 0.3-0.5M HCl solution (5-10 times the weight) at a temperature of 25°C for 2-3 hours, and then rinsing it with clean water; soaking the fish skin in n-hexane for 5-6 hours to remove fat, and washing it to obtain defatted fish skin.

[0035] Step S2: Compound enzymatic hydrolysis: The crude collagen gel solution is subjected to acidic enzymatic hydrolysis and alkaline enzymatic hydrolysis twice in sequence, and the steps are as follows:

[0036] 1) First acid enzymatic hydrolysis:

[0037] The crude collagen gel solution obtained above (preferably having a solid content of about 5-15 wt%) is adjusted to a pH of 6-6.8 with a hydrochloric acid solution, and then a mixed enzyme of papain and flavor protease is added under stirring for enzymatic hydrolysis to obtain an acidic hydrolyzate. The enzymatic hydrolysis temperature is 55-60° C. and the enzymatic hydrolysis time is 5-10 hours. The enzyme dosage of papain is 40-60 U / g (based on the wet weight of fish scales), and the enzyme dosage of flavor protease is 20-30 U / g (fish scales).

[0038] 2) Secondary alkaline enzymatic hydrolysis:

[0039] After adjusting the pH of the obtained acidic enzymatic hydrolysate to 7.4-7.6 with sodium hydroxide solution, a mixed enzyme of collagenase and alkaline protease is added for stirring and enzymatic hydrolysis. The enzymatic hydrolysis temperature is 37-45°C and the enzymatic hydrolysis time is 6-12 hours. The amount of collagenase added is 40-50 U / g (fish scale, wet weight, the same below), and the amount of alkaline protease added is 20-30 U / g (fish scale). The collagenase is selected from at least one of Clostridium histolyticum collagenase and Vibrio alginolyticus collagenase. The alkaline protease is selected from Properase E or Bacillus subtilis alkaline protease, preferably Properase E alkaline protease.

[0040] 3) After enzymatic hydrolysis, the enzyme was inactivated by heating in a 100°C water bath for 10-15 minutes, and then centrifuged at 4°C using a high-speed continuous centrifuge (10-13K RPM) for 15-20 minutes. The supernatant was removed to obtain a crude enzymatic hydrolyzed collagen peptide solution.

[0041] Step S3: Preliminary ultrafiltration separation:

[0042] The obtained enzymatically hydrolyzed collagen peptide crude solution is subjected to ultrafiltration filtration; wherein the enzymatically hydrolyzed collagen peptide solution is passed through an ultrafiltration membrane with a molecular weight cutoff of 2000-3000 Daltons (abbreviated as D or Dal) to remove macromolecular impurities, thereby obtaining an ultrafiltered enzymatically hydrolyzed collagen peptide solution.

[0043] Ultrafiltration treatment can separate incompletely hydrolyzed macromolecular collagen from collagen peptide molecules with a higher degree of hydrolysis; wherein the retained incompletely hydrolyzed macromolecular collagen is recovered and added to the raw materials of the aforementioned enzymatic hydrolysis step to continue the enzymatic hydrolysis treatment.

[0044] This step does not require desalting or adding activated carbon for decolorization and deodorization.

[0045] Step S4: Nonspecific separation based on magnetic microsphere media

[0046] 1) The ultrafiltration-treated enzymatic collagen peptide solution is moderately concentrated and then loaded onto a separation column that has been rinsed and equilibrated with deionized water. The separation column is sequentially segmented from top to bottom and filled with mesoporous silica filler and magnetic microsphere filler. The magnetic microsphere filler consists of segmented polymethacrylate magnetic microsphere filler and PVA magnetic microsphere filler (there is no limit to the order in which the two magnetic microspheres are loaded; in addition, the two microsphere fillers can be mixed and loaded, i.e., PMMA / PVA mixed microsphere filler is used as the lower section of the separation column), wherein the height of the upper mesoporous silica filler does not exceed 30% of the total filler height, preferably 10-30%, and is loaded at the top; preferably, the amount of polymethacrylate magnetic microsphere filler and PVA magnetic microsphere filler is 0.5-2:1, more preferably 1:1, that is, equal loading.

[0047] Among them, the polymethacrylate magnetic microspheres are preferably polymethyl methacrylate magnetic microspheres (PMMA), more preferably microspheres with surface amino or carboxyl modified (to enhance the hydrogen bonding ability with the terminal amino acid residues of collagen tripeptide), preferably those with a particle size of 10-50 microns and a pore size of less than 200 nm.

[0048] The polyvinyl alcohol magnetic microspheres preferably have a particle size of 20-50 microns and a surface hydroxyl value of not less than 10 mmol / g.

[0049] In this step, although the polymethacrylate magnetic microsphere filler and the PVA magnetic microsphere filler can be mixed and packed, it is preferred to adopt segmented packing to facilitate the separation of the fillers in the subsequent regeneration treatment stage.

[0050] The mesoporous silica particles have a diameter of 1-10 microns, preferably 1-5 microns. When passing through this filler, the macromolecular peptides and salt-like non-peptide components in the solution can pass through more quickly than the collagen tripeptide, which can easily enter the pore structure. This allows for initial separation from the collagen tripeptide, facilitating further separation of the microsphere filler.

[0051] 2) First, elution with 2-3 column volumes of ethanol-water (10-20% by volume) to remove salts and non-peptide small molecule impurities is performed. Then, elution is performed sequentially with 8-10 column volumes of deionized water and 3-8 wt% dilute ammonia (3-5 column volumes) as eluents until the eluent is substantially free of polypeptide or amino acid components. The eluents containing the collagen tripeptide component are collected and combined, and the ammonia component is evaporated under reduced pressure and concentrated to obtain a crude collagen tripeptide solution after nonspecific separation using the silica / magnetic microsphere composite filler.

[0052] The remaining eluate containing macromolecular peptides is subjected to reduced pressure evaporation of the solvent and concentrated, which can be used as a recovered mother liquor and mixed with the next batch of raw materials to be separated for recycling. The vacuum degree of reduced pressure concentration can be, for example, -0.9 MPa or above.

[0053] Among them, the collagen tripeptide component in the eluate is detected by HPLC (HPLC chromatographic analysis method and conditions are known, and this application refers to WO2016 / 076647). Specifically, the collagen tripeptide standard (glycine-proline-hydroxyproline) is used as a reference for the collagen tripeptide standard. In this step, the sample is dried and then analyzed by liquid chromatography. The purity or content of the obtained collagen tripeptide product is above 60%.

[0054] In this step, the intermolecular forces such as hydrogen bonding between the surface groups such as hydroxyl and amino groups on the surface of the magnetic microspheres and the terminal amino acid residues of the collagen tripeptide short peptides are stronger (the smaller the peptide, the higher the proportion of amino and carboxyl groups), and the small molecule peptides are more likely to enter the internal gaps of the microspheres, resulting in a slower elution rate, thereby separating the collagen tripeptide from the large molecule peptides. For example, the richer the surface hydroxyl groups of the polyvinyl alcohol magnetic microspheres, the stronger the hydrogen bonding force with the collagen tripeptide small peptides.

[0055] In addition, compared with the direct use of non-magnetic microspheres, magnetic microspheres have a more uniform particle size distribution and pore structure, and the microspheres are relatively large in size (compared to silica gel chromatography), and the separation rate is faster; at the same time, the metal chelation / coordination force of the magnetic metal particles and the rich hydroxyl, amino, carboxyl and other groups on the surface of the microspheres make the interaction with polypeptide molecules of different sizes different in strength, making polypeptide separation more efficient and faster than simple adsorption chromatography separation.

[0056] The polyvinyl alcohol or polymethacrylate magnetic microspheres can be prepared according to general methods in the art (the preparation of magnetic microspheres is a known technology in the art), or can be commercially obtained.

[0057] Exemplarily, the preparation process of PVA magnetism is as follows: heat and dissolve polyvinyl alcohol in water and stir to form a uniform solution; weigh a mixed raw material of ferrous chloride and ferric chloride with a molar ratio of 1:2, dissolve it in an appropriate amount of deionized water, stir evenly and add it to the above-mentioned PVA solution, start stirring, add an appropriate amount of liquid paraffin or Tween and stir evenly; add concentrated ammonia water in batches under water bath heating conditions, and continue stirring to react after the addition is completed; after the reaction is completed, cool to room temperature, centrifuge or magnetically separate the microspheres, wash the microsphere particles with deionized water, and vacuum dry to obtain magnetic microspheres with polyvinyl alcohol coated on the surface.

[0058] Surface-modified polymethacrylate magnetic microspheres can also be prepared using methods known in the art for surface modification of microspheres, or can be commercially obtained. For example, surface-amino-modified polymethacrylate magnetic microspheres can be prepared by dispersing polymethyl methacrylate magnetic microspheres, a lower alkylamine such as ethylamine / diamine, and an acrylic silane coupling agent in an alcohol solvent, stirring and heating the mixture in a water bath for 6-12 hours, and then washing the resulting microspheres. For example, the mass ratio of magnetic microspheres to fatty amine can be 10:1-3.

[0059] For example, polymethyl methacrylate magnetic microspheres, amines and acrylate coupling agents are dispersed in methanol and stirred for 12 hours. After the reaction, the microspheres are separated and washed with alcohol and deionized water in sequence to obtain amino-modified polymethacrylate magnetic microspheres, which are dried and set aside.

[0060] Step S5: Gradient solid phase extraction purification using molecular imprinting method

[0061] The non-specifically separated crude collagen tripeptide solution is further selectively purified and separated using a molecular imprinting method based on different template molecules. This step includes vacuum concentrating the non-specifically separated crude collagen tripeptide solution and then loading it into a separation column containing a molecular imprinting polymer prepared based on different template molecules, and then eluting.

[0062] The different template molecules include: at least two, preferably three or more, of the following: a collagen tripeptide template molecule with a terminal group of glycine (Gly), a dipeptide template molecule with a terminal group of glycine (Gly), and a collagen tripeptide characteristic amino acid (glycine, hydroxyproline) template molecule.

[0063] Specifically, the collagen tripeptide template molecule with Gly terminal group is selected from at least one of glycine-proline-hydroxyproline (Gly-Pro-Hyp) and glycine-hydroxyproline-proline (Gly-Hyp-Pro), and preferably contains at least Gly-Pro-Hyp.

[0064] The dipeptide template molecule having a terminal group of glycine (Gly) is selected from at least one of Gly-Pro and Gly-Hyp, and preferably contains at least Gly-Pro.

[0065] The characteristic amino acids of the collagen tripeptide are glycine and / or hydroxyproline, and preferably contain at least Hyp.

[0066] Preferably, the collagen tripeptide template molecule is a combined template molecule of Gly-Pro-Hyp and Gly-Hyp-Pro, wherein the molar content of the Gly-Pro-Hyp template molecule is not less than 50% (preferably not less than 60%), which is recorded as template molecule A.

[0067] Preferably, the dipeptide template molecule is a combination template molecule of Gly-Pro and Gly-Hyp; wherein the molar content of the Gly-Pro template molecule is not less than 50% (preferably not less than 60%), which is recorded as template molecule B.

[0068] Preferably, the characteristic amino acids of the collagen tripeptide are a template molecule composed of Gly and Hyp, wherein the molar content of the Gly template molecule is not less than 30%, which is recorded as template molecule C.

[0069] The molecularly imprinted polymer is prepared by thermal polymerization using the aforementioned collagen tripeptide, dipeptide, and characteristic amino acids as template molecules in the presence of functional monomers and a cross-linking agent. The molecularly imprinted polymer can be prepared according to general methods in the art, and its preparation method is well known in the art.

[0070] Specifically, the above-mentioned template molecules A, B, and C are respectively used as template molecules, and acrylamide functional monomers are used to carry out thermal polymerization reaction in the presence of an acrylate crosslinker and an initiator to obtain corresponding polymers (which can be recorded as polymers A, B, and C). The template molecules in the polymers are hydrolyzed or eluted to obtain the collagen tripeptide derivative molecularly imprinted polymer.

[0071] For example, the collagen tripeptide template molecules (A, B, C) are stirred and mixed in an organic solvent, and functional monomers such as acrylamide or methacrylic acid are added; a crosslinker (for example, N,N'-methylenebisacrylamide or ethylene glycol dimethacrylate EGDMA) and an appropriate amount of initiator azobisisobutyronitrile AIBN or ammonium persulfate are then added. After ultrasonic mixing, the reaction system is sealed under a nitrogen atmosphere and stirred at 60°C for 16-24 hours; after the reaction is completed, the reaction is filtered, washed thoroughly with deionized water and acetone in sequence, and dried. The polymer is then sieved to control the uniformity of the particle size, and then treated with hydrochloric acid and saturated sodium chloride solution in sequence to remove the template molecules (template molecules A and B). The residual template molecules, functional monomers, and other organic substances are then thoroughly soaked and washed with acetonitrile-water solution and anhydrous methanol. Finally, the resulting polymer microspheres are vacuum dried to obtain molecularly imprinted polymers.

[0072] Specifically, the steps of gradient solid phase extraction purification using molecular imprinting method are as follows:

[0073] 1) The molecularly imprinted polymer microspheres prepared from template molecules A, B, and C are loaded onto the column in order from top to bottom (preferably, the volume of the molecularly imprinted polymer prepared from template molecules A and B is not less than 50% of the total volume of the column), the column is equilibrated with deionized water, and then the concentrated crude collagen tripeptide solution is loaded (preferably the peptide content is controlled to be no more than 0.1 g / ml); then, 1-3 column volumes of deionized water are used to elute and remove impurities, such as components such as large molecular peptides that cannot be specifically adsorbed.

[0074] The impurity-removed liquid obtained in this step can be recycled as mother liquor in this step after evaporation and concentration, or combined with the mother liquor in the previous step and recycled as raw material.

[0075] The amount of the polypeptide in this step should preferably not exceed 50% of the maximum adsorption capacity of the molecularly imprinted polymer; preferably, the actual amount of the molecularly imprinted polymer added far exceeds the theoretical amount.

[0076] 2) Then, 5-10 column volumes of deionized water, 3-5 column volumes of ethanol-water solution (preferably 10-20% volume fraction), and 1-5 wt% dilute ammonia solution are sequentially used to fully elute until there is substantially no amino acid and peptide components, and the collected collagen tripeptide eluates are combined.

[0077] 3) The combined eluates are filtered through a 0.22 μm filter membrane, concentrated by evaporation under reduced pressure to remove the organic solvent and ammonia components, and the solid content is controlled. The eluates are then spray-dried or freeze-dried to obtain a composition having a high content of collagen tripeptide, wherein the content of small molecule collagen peptides with a molecular weight of no more than 1 kDa is no less than 95%, and the content of collagen tripeptide is no less than 80%. The composition is dried, sterilized by irradiation, and then packaged.

[0078] The present invention uses a molecular imprinting method that combines collagen tripeptide, dipeptide molecular templates and characteristic amino acid molecular imprinting polymers, which can not only achieve rapid separation and purification of collagen tripeptide and its derivatives, but also significantly improve the purification efficiency of small molecule peptides including collagen tripeptide through specific adsorption by multi-layer chromatography. This method of gradient solid-phase extraction using segmented fillers, compared to the molecular imprinting method of a single template molecule, makes the distribution of specific binding sites of the collagen tripeptide molecules of the polymer filler in the filler system more extensive. The specific gradient adsorption method of the filler polymer with strong top and weak bottom not only avoids the problem of insufficient adsorption and binding strength of molecular imprinting for polypeptide molecules, but is also more conducive to the synchronous elution of collagen tripeptide molecules, so that the collagen tripeptide components are eluted relatively concentratedly, which is beneficial to improving the purity of the obtained collagen tripeptide.

[0079] In a second aspect, the present invention provides a collagen tripeptide composition product obtained based on the above method, characterized in that the collagen tripeptide content is not less than 80%, and the content of peptides with a molecular weight greater than 1 KDa does not exceed 5%.

[0080] In a third aspect, the present invention also provides applications of the collagen tripeptide composition product in the fields of beauty, cosmetics or skin care, medicine, and food.

[0081] The present invention has the following beneficial effects compared to the prior art:

[0082] 1) After performing two enzymatic hydrolysis on the pretreated collagen raw material, the present invention uses a specific system of magnetic microsphere separation and molecular imprinting polymer microsphere medium solid phase extraction to purify and separate it. In particular, the molecular imprinting method, which combines characteristic amino acids, dipeptide template molecules, and tripeptide template molecules designed based on the molecular structure of collagen tripeptide, has excellent selectivity for collagen tripeptides with glycine end groups and rich in hydroxyproline. This overcomes the disadvantage of existing purification methods that cannot obtain high-purity collagen tripeptides in batches through ion exchange and column chromatography; the method of the present invention can significantly improve the separation efficiency and separation purity of the product, and the collagen tripeptide content reaches 80% or even more than 85%.

[0083] 2) The present invention uses nonspecific adsorption separation based on magnetic microspheres. Compared with large molecular peptides, smaller numbers of small molecular peptides, such as dipeptides and collagen tripeptides, have a relatively high proportion of surface exposed amino, carboxyl / hydroxyl and other end groups, and thus have stronger hydrogen bonding forces with the surface groups of magnetic microspheres. This difference in the intermolecular forces between large and small peptide molecules and the surface groups of microspheres makes the separation of large quantities of collagen tripeptides more significant.

[0084] 3) The present invention uses gradient solid-phase extraction of different molecularly imprinted polymer combination fillers to enable layered specific adsorption of collagen tripeptide molecules, significantly improving the purification efficiency; while other peptide molecules that do not contain the characteristic amino acids of collagen tripeptide, especially relatively large peptide fragments, are easily eluted, thereby easily separating them from the collagen tripeptide derivatives.

[0085] The present invention discovered that, under the same conditions, the molecularly imprinted polymer (MIP) prepared from template molecule A in the form of a collagen tripeptide molecule exhibited the highest selectivity and strongest binding for collagen tripeptide derivatives (with a relatively slow elution rate), making it suitable for upper column packing. The MIP prepared from template molecule B in the form of a dipeptide molecule exhibited inferior selectivity and binding for collagen tripeptide derivatives, while the MIP prepared from template molecule C in the form of characteristic amino acids (glycine and / or hydroxyproline) exhibited relatively poor selectivity and binding for collagen tripeptide derivatives and was easily eluted, making it suitable for lower column packing. Larger peptide molecules (over 1000 Da) containing a large number of amino acid residues exhibited poor specific adsorption capacity when combined with the MIP filler and were easily eluted, thereby significantly reducing the content of large peptides over 1000 Da in the final product obtained through separation and purification (generally no more than 5%). DETAILED DESCRIPTION

[0086] The present invention is described in detail below through specific examples, but these embodiments do not constitute any form of limitation to the protection scope of the present invention.

[0087] Preparation Example 1

[0088] Preparation of surface amino-modified polymethacrylate magnetic microspheres

[0089] Take 100 g of polymethyl methacrylate magnetic microspheres, 12 g of ethylenediamine and 30 g of trimethoxysilyl-substituted acrylate coupling agent and disperse them in 1 L of methanol. The closed reactor is slowly stirred and reacted in a water bath at 45°C for 12 hours. After the reaction, the microspheres are filtered and separated, and then washed thoroughly with methanol and deionized water in sequence to obtain amino-modified polymethacrylate magnetic microspheres, which are dried and set aside.

[0090] Preparation Example 2

[0091] Preparation of molecularly imprinted polymers

[0092] 1) 1 mmol of collagen tripeptide template molecule A (a composite template molecule with a molar ratio of Gly-Pro-Hyp:Gly-Hyp-Pro of 8:2) was stirred in 50 ml of a mixed solvent of DMSO and chloroform (equal volume ratios). Then, 10 mmol of the functional monomer acrylamide, 40 mmol of ethylene glycol dimethacrylate, and 680 mg of azobisisobutyronitrile were added. After ultrasonic mixing, the nitrogen atmosphere was replaced, and the reaction system was sealed and stirred at 60°C and 200 rpm for 24 h. After completion of the reaction, the polymer was filtered, washed thoroughly with deionized water and acetone, and dried. The polymer was then sieved to control uniformity. The peptide template was then hydrolyzed with 5 mol / L hydrochloric acid and washed with saturated sodium chloride solution to remove the template. Residual template molecules, functional monomers, and other organic matter were then washed thoroughly with a 30% (volume fraction) acetonitrile-water solution and anhydrous methanol. The resulting polymer microspheres were vacuum dried at 50°C to obtain molecularly imprinted polymers with an average particle size of approximately 50 μm.

[0093] 2) In addition to replacing template molecule A with an equal molar amount of template molecule B

[0094] The molecularly imprinted polymer B was prepared by the same operation as in step 1) except for the combined template molecule with a molar ratio of Gly-Pro:Gly-Hyp=7:3.

[0095] 3) Molecularly imprinted polymer C was prepared by the same operation as in step 1) except that template molecule A was replaced by an equimolar amount of template molecule C (combined template molecule with a molar ratio of Gly:Hyp=5:5) and hydrochloric acid hydrolysis was omitted.

[0096] Example 1

[0097] The steps for preparing high-purity collagen tripeptide are as follows:

[0098] Step S1: Pretreatment of collagen raw materials

[0099] 5 kg of fish scales (from carp) were cleaned, dried, and crushed to approximately 40 mesh. The scales were then soaked in 50 L of water at 50°C for 6 hours, then filtered and drained. The resulting solid was soaked in 30 L of 0.2 M HCl solution at room temperature for 5 hours, then rinsed with clean water and filtered until clean. The scales were placed in a heated kettle with stirring, and 25 kg of deionized water was added. The scales were heated at 100°C under sealed stirring for 8 hours to obtain a scale glue solution. The resulting scale glue solution was filtered while hot to remove solid scale residue and other impurities. The filtrate was cooled to room temperature to obtain a crude collagen glue solution.

[0100] Step S2: Compound enzymatic hydrolysis: The crude collagen gel solution is subjected to acidic enzymatic hydrolysis and alkaline enzymatic hydrolysis in sequence, as follows:

[0101] 1) The crude collagen gel solution obtained is evaporated and concentrated to about 11 kg, the pH of the solution is adjusted to 6.5-6.6 with hydrochloric acid, and a mixture of papain and flavor protease is added under stirring for enzymatic hydrolysis to obtain an acidic enzymatic hydrolyzate. The enzymatic hydrolysis temperature is 55-56° C. and the enzymatic hydrolysis time is 6 hours to obtain an acidic enzymatic hydrolyzate. The amount of papain used is 50 U / g (based on the wet weight of fish scales, the same below), and the amount of flavor protease used is 25 U / g.

[0102] 2) After adjusting the pH of the obtained acidic enzymatic hydrolysate to 7.5-7.6 with a sodium hydroxide solution, a mixture of collagenase (Clostridium histolyticum collagenase) and alkaline protease (Properase E) was added and stirred for enzymatic hydrolysis at a temperature of 40-42° C. for 8 hours; wherein the amount of collagenase added was 50 U / g (fish scale) and the amount of alkaline protease added was 30 U / g (fish scale).

[0103] 3) After enzymatic hydrolysis, the enzyme was inactivated by heating at 100°C for 15 minutes, and then the mixture was centrifuged in batches at 4°C for 20 minutes using a high-speed continuous centrifuge (12,000 RPM). The supernatants were removed and combined to obtain a crude enzymatic collagen peptide solution.

[0104] Step S3: The crude enzymatically hydrolyzed collagen peptide solution prepared above is subjected to ultrafiltration; wherein the enzymatically hydrolyzed collagen peptide solution is passed through an ultrafiltration membrane with a molecular weight cutoff of 2000D to remove macromolecular impurities, and about 9.8 kg of ultrafiltration-treated enzymatically hydrolyzed collagen peptide solution is obtained.

[0105] Step S4: Nonspecific separation based on magnetic microsphere media

[0106] 1) 1 L of the above-mentioned ultrafiltered enzymatic collagen peptide solution was loaded in batches onto a 125 cm separation column rinsed and equilibrated with deionized water. The separation column was sequentially loaded with mesoporous silica filler (particle size of about 3-5 microns) and composite magnetic microsphere filler from top to bottom. The composite magnetic microsphere filler was composed of equal weight ratios of amino-surface-modified polymethacrylate magnetic microsphere filler (PMMA) and PVA magnetic microsphere filler (particle size 30-40 microns) prepared in the above preparation example, which were divided into two sections (there is no restriction on the order of loading the two magnetic microspheres, that is, polymethacrylate magnetic microsphere filler can also be used as the lower section filler), wherein the height of the upper mesoporous silica filler is one-fifth of the total filler height.

[0107] 2) First, elution was performed with 2.5 column volumes of 10% ethanol-water solution to remove salts and non-peptide small molecule impurities. Then, elution was performed with 8 column volumes of deionized water and approximately 3 column volumes of dilute ammonia (5 wt%), respectively, at a flow rate of 3 BV / h, until the eluents were substantially free of peptide or amino acid components. The eluents containing the collagen tripeptide fraction were pooled, and the ammonia fraction was evaporated under reduced pressure and concentrated to obtain a crude collagen tripeptide solution after nonspecific separation using the silica / magnetic microsphere composite filler.

[0108] The remaining eluate was subjected to reduced pressure evaporation of the solvent, concentrated, and used as a recovered mother liquor to be mixed with the next batch of raw materials to be separated for recycling. The vacuum degree of the reduced pressure concentration was -0.9 MPa.

[0109] Among them, based on the HPLC chromatogram of the collagen tripeptide standard (glycine-proline-hydroxyproline) as a reference, the collagen tripeptide component in the eluate was monitored and analyzed by HPLC, and then the eluate containing the collagen tripeptide component was collected. The HPLC chromatographic analysis conditions refer to WO2016 / 076647 (i.e., the mobile phase is 10mM Tris-Cl, pH 7.4, 5mM CaCl2, and the flow rate is 0.5ml / min). In this step, the relative content of collagen tripeptide in the crude collagen tripeptide solution was measured to be about 68%.

[0110] Step S5: Gradient solid phase extraction purification using molecular imprinting method, the steps are as follows:

[0111] 1) The molecularly imprinted polymer microspheres A, B, and C prepared in the above preparation example were packed into a column in order from top to bottom to a packing height of approximately 80 cm (wherein the packing volume ratio of molecularly imprinted polymers A, B, and C was 4:4:2, respectively). After equilibration of the column with deionized water, the concentrated crude collagen tripeptide solution (content approximately 0.1 g / ml) obtained by nonspecific separation was loaded in batches. The solution was then eluted with 3 column volumes of deionized water to remove impurities. The impurity-removed solution was concentrated by evaporation and recovered as the mother liquor for recycling.

[0112] 2) Elution was carried out in sequence using 5 column volumes of deionized water and 3 column volumes of ethanol aqueous solution (20% volume fraction), and finally eluted with 5wt% dilute ammonia water until it was substantially free of amino acids and peptide components, and the eluate components were collected. The eluates were combined and filtered through a 0.22μm filter membrane, evaporated and concentrated under reduced pressure to remove alcohol solvents and ammonia components to obtain a purified collagen tripeptide solution. Furthermore, the obtained concentrated solution was spray-dried to obtain a solid composition with a high content of collagen tripeptide. The samples were analyzed by HPLC content, and the results showed that the content of small molecule collagen peptides with a molecular weight of less than 1KDa was about 96.4%, the content of collagen tripeptide (Gly-XY) was 88.3%, and the content of glycine-proline-hydroxyproline (GPH) was about 18.7%.

[0113] Example 2

[0114] Preparation steps S1-S3 are the same as in Example 1 (i.e., the enzymatic collagen peptide solution subjected to ultrafiltration treatment in step S3 of Example 1 is used). Steps S4-S5 are as follows:

[0115] Step S4: Nonspecific separation

[0116] 1) 1 L of ultrafiltered enzymatic collagen peptide solution was loaded in batches onto a 125 cm separation column rinsed and equilibrated with deionized water. The separation column was sequentially loaded with mesoporous silica filler and composite magnetic microsphere filler from top to bottom. The composite magnetic microsphere filler was composed of a direct mixture of amino-modified polymethacrylate magnetic microsphere filler (PMMA) and PVA magnetic microsphere filler in equal weight ratios, wherein the height of the mesoporous silica filler at the top was 30% of the total filler height.

[0117] 2) Elution with 2 column volumes of ethanol-water (10% by volume) to remove impurities, followed by elution with 10 column volumes of deionized water, and elution with dilute ammonia (5 wt%) until the peptide components are essentially eliminated, at an elution flow rate of 2.5 BV / h; the eluate containing the collagen tripeptide component is collected and combined, the alcohol solvent and ammonia component are evaporated under reduced pressure, and concentrated to obtain a crude collagen tripeptide solution after nonspecific separation. The remaining eluate is subjected to reduced pressure evaporation of the solvent, concentrated, and then used as a recovered mother liquor to be mixed with the next batch of raw materials to be separated for recycling.

[0118] By HPLC sampling analysis, the collagen tripeptide content in this step was measured to be approximately 69.6% (it may be that the mixing of different microspheres makes the pores smaller, and the types of surface groups of the mixed microsphere fillers are more complex, and the adsorption force between the polypeptide molecules is enhanced, resulting in an increase in the purity of the tripeptide, but it also makes the elution incomplete, resulting in additional increase in product column loss, and makes the subsequent separation and recovery of the two microspheres difficult, so this mixed filler method is not the preferred method).

[0119] Step S5: Gradient solid phase extraction purification using molecular imprinting method, the steps are as follows:

[0120] 1) The molecularly imprinted polymer microspheres A, B, and C prepared in the above preparation example were packed into a column in order from top to bottom to a packing height of approximately 80 cm (wherein the packing volume ratio of molecularly imprinted polymers A, B, and C was 5:3:2, respectively). After equilibration of the column with deionized water, the concentrated crude solution of nonspecifically separated collagen tripeptide (approximately 0.12 g / ml) was loaded in batches. The solution was then eluted with 3 column volumes of deionized water to remove impurities. The impurity-removed solution was evaporated and concentrated, and then recovered as the mother liquor for recycling.

[0121] 2) Elution was performed sequentially with 6 column volumes of deionized water and 3.5 column volumes of ethanol aqueous solution (20% volume fraction), and finally with 5wt% dilute ammonia water until the solution was substantially free of amino acids and peptide components, and the eluate components were collected. The eluates were combined and filtered through a 0.22μm filter membrane, evaporated and concentrated under reduced pressure to remove the alcohol solvent and ammonia components to obtain a purified collagen tripeptide solution. The resulting concentrated solution was spray-dried to obtain a solid composition with a high content of collagen tripeptide. Sampling was analyzed by HPLC, and the results showed that the collagen tripeptide (Gly-XY) content was 86.8%, of which the GPH content was approximately 20.1%.

[0122] The results show that increasing the proportion of molecularly imprinted polymer microspheres A filler is beneficial to increasing the specifically adsorbed GPH content to a certain extent, but the relative proportions of other fillers B and C are reduced, resulting in a decrease in the overall collagen tripeptide adsorption performance, which is not conducive to increasing the total content of collagen tripeptide.

[0123] Comparative Example

[0124] Steps S1-S2 are the same as those in Example 1 (i.e., the crude enzymatic collagen peptide solution in step S2 of Example 1 is used). The remaining steps are as follows:

[0125] Step S3: 1 L of the crude enzymatic collagen peptide solution was decolorized with activated carbon and filtered; the filtrate was then applied to a macroporous adsorption resin column, and salts and small molecular organic impurities were first eluted with 3 column volumes of 20% ethanol; then the elution was thoroughly eluted with deionized water and 5 wt% ammonia water, and the deionized water eluate and the ammonia eluate were collected and then evaporated and concentrated;

[0126] Step S4: The eluate is subjected to nanofiltration: a nanofiltration membrane with a molecular weight cutoff of 1000D is first used, and the permeate is concentrated through a nanofiltration membrane with a molecular weight cutoff of 200D (membrane operating pressure 0.25MPa) to obtain a collagen tripeptide solution, which is freeze-dried to obtain a fish scale collagen tripeptide composition, wherein the collagen tripeptide (CTP) content is about 70.1% (wherein the GPH content is about 9.8%), and the yield (peptide powder / fish scale, based on dry weight) is about 11.6% (compared to Example 1, a decrease of about 36%, which may be due to the loss of activated carbon decolorization step and the loss of nonspecific adsorption of macroporous resin, as well as the loss of nanofiltration membrane step).

[0127] The above embodiments do not limit the technical solutions of the present invention. Those skilled in the art may modify the technical solutions described in the above embodiments, and these modifications or replacements do not depart from the scope of the technical solutions of the present invention.

Claims

1. A method for separating and purifying collagen tripeptides from a collagenase hydrolysate, wherein the collagenase hydrolysate is obtained by pre-treating the collagen raw material and then hydrolyzing it with a composite enzyme, wherein the hydrolysis comprises at least one collagenase; characterized in that: The following steps are involved: 1) The collagenase hydrolysate is ultrafiltered and then subjected to nonspecific adsorption separation, including: The enzymatically hydrolyzed collagen peptide solution subjected to ultrafiltration is loaded onto a separation column, which is sequentially filled with mesoporous silica fillers and magnetic microsphere fillers from top to bottom in sections. The magnetic microsphere fillers are composed of polymethacrylate magnetic microsphere fillers and PVA magnetic microsphere fillers, wherein the height of the upper mesoporous silica filler does not exceed 30% of the total filler height; the polymethacrylate magnetic microspheres are amino-surface-modified polymethyl methacrylate magnetic microspheres with a particle size of 10-50 μm; the PVA magnetic microspheres have a particle size of 20-50 μm; and the mesoporous silica has a particle size of 1-10 μm. 2) Gradient solid phase extraction purification using molecular imprinting: The collagen peptides obtained by nonspecific adsorption separation are loaded onto molecularly imprinted polymer separation columns prepared with different template molecules for specific separation and purification; The collagen tripeptide template molecule with a Gly terminal group is selected from at least one of Gly-Pro-Hyp and Gly-Hyp-Pro, the dipeptide template molecule with a Gly terminal group is selected from at least one of Gly-Pro and Gly-Hyp, and the collagen tripeptide characteristic amino acid template molecule is selected from at least one of glycine and hydroxyproline; The above-mentioned different template molecules are respectively used to carry out thermal polymerization reaction using acrylamide functional monomers in the presence of an acrylate crosslinker and an initiator to obtain corresponding polymers, and the template molecules in the polymer are removed to obtain the corresponding molecularly imprinted polymers; the crosslinker is selected from N,N'-methylenebisacrylamide or ethylene glycol dimethacrylate; the initiator is selected from azobisisobutyronitrile or ammonium persulfate; the thermal polymerization reaction conditions are: the reaction system is sealed under a nitrogen atmosphere, and the reaction is stirred at 60°C for 16-24 hours.

2. The method according to claim 1, characterized in that The method for obtaining the collagen enzymatic hydrolysate by pre-treating the collagen raw material and then hydrolyzing it with a composite enzyme comprises the following specific steps S1-S3: Step S1: preparing an enzymatic hydrolyzed collagen peptide solution: pre-treating a collagen raw material to obtain a crude collagen gel; wherein the collagen raw material is selected from fish scales, defatted fish skin, or a mixture of the two; Step S2: subjecting the obtained crude collagen gel solution to acidic enzymatic hydrolysis and alkaline enzymatic hydrolysis twice in sequence, wherein the enzymatic hydrolysis comprises at least collagenase; Step S3: ultrafiltration is performed on the obtained crude solution of enzymatically hydrolyzed collagen peptides; wherein the enzymatically hydrolyzed collagen peptide solution is filtered through an ultrafiltration membrane with a molecular weight cutoff of 2000-3000D to obtain an ultrafiltration-treated enzymatically hydrolyzed collagen peptide solution.

3. The method according to claim 1, characterized in that The mass ratio of polymethacrylate magnetic microsphere filler and PVA magnetic microsphere filler in the magnetic microsphere filler is 0.5-2:

1.

4. The method according to claim 1, wherein The collagenase hydrolysate is subjected to ultrafiltration and then to nonspecific adsorption separation, and the specific operation is as follows: 1) The ultrafiltration-treated enzymatically hydrolyzed collagen peptide solution is concentrated and loaded onto a separation column, wherein the separation column is sequentially packed with mesoporous silica filler and magnetic microsphere filler in sections from top to bottom, wherein the magnetic microsphere filler comprises polymethacrylate magnetic microsphere filler and PVA magnetic microsphere filler packed in sections, wherein the height of the upper mesoporous silica filler does not exceed 30% of the total filler height and is packed in the upper section; the ratio of the polymethacrylate magnetic microsphere filler to the PVA magnetic microsphere filler is 1:1; 2) First, use a 10-20% volume fraction of ethanol aqueous solution to remove impurities, and then use deionized water and 3-8wt% dilute ammonia water as eluents to elute until the eluent is essentially free of polypeptide or amino acid components, collect and combine the eluent containing the collagen tripeptide component, evaporate the ammonia component under reduced pressure and concentrate to obtain a crude collagen tripeptide solution after nonspecific separation.

5. The method according to claim 1, wherein The molecular imprinting method is used for gradient solid phase extraction purification, and the specific operation is as follows: 1) sequentially packing a column with a collagen tripeptide template molecule, a dipeptide template molecule, and a molecularly imprinted polymer microsphere prepared from a collagen tripeptide characteristic amino acid template molecule, wherein the column packing volume of the molecularly imprinted polymer prepared from the collagen tripeptide template molecule and the dipeptide template molecule is not less than 50% of the total column packing volume; loading a crude collagen tripeptide solution; and eluting with 1-3 column volumes of deionized water to remove impurities; 2) eluting with deionized water, 10-20% volume fraction ethanol aqueous solution, and 1-5wt% dilute ammonia water in sequence until the solution is substantially free of amino acids and peptide components, and combining the collagen tripeptide eluates; 3) The eluate is filtered through a 0.22 μm filter membrane, concentrated by evaporation under reduced pressure to remove the organic solvent and ammonia components, and then spray-dried or freeze-dried to obtain a composition with a high content of collagen tripeptide, wherein the content of collagen tripeptide is not less than 80%.

6. The method according to claim 2, characterized in that The specific operations of step S2 are as follows: 1) Acidic enzymatic hydrolysis: After adjusting the pH of the crude collagen solution to 6-6.8 with hydrochloric acid solution, a mixture of papain and flavor protease is added under stirring to perform enzymatic hydrolysis to obtain an acidic enzymatic hydrolyzate. The enzymatic hydrolysis temperature is 55-60° C. and the enzymatic hydrolysis time is 5-10 hours. The amount of papain used is 40-60 U per gram of fish scale, and the amount of flavor protease used is 20-30 U per gram of fish scale, based on the weight of the fish scale; 2) adjusting the pH of the obtained acidic enzymatic hydrolysate to 7.4-7.6 with sodium hydroxide solution, adding a mixture of collagenase and alkaline protease and stirring for enzymatic hydrolysis at a temperature of 37-45° C. for 6-12 hours; the collagenase is selected from at least one of Clostridium histolyticum collagenase or Vibrio alginolyticus collagenase, and the alkaline protease is selected from at least one of Properase E or Bacillus subtilis alkaline protease; wherein the amount of collagenase added is 40-50 U per gram of fish scale, and the amount of alkaline protease added is 20-30 U per gram of fish scale; 3) After enzymatic hydrolysis, the enzyme is inactivated by heating at 100° C. for 10-15 minutes, and then centrifuged at 4° C. for 15-20 minutes using a high-speed continuous centrifuge to obtain a crude enzymatic hydrolyzed collagen peptide solution.

7. The method according to claim 2, characterized in that The specific operations of step S1 are as follows: Fish scales are used as raw materials and are directly crushed and pulped by adding water; or the fish scales are washed, dried, crushed and sieved, and then processed according to the following steps: the crushed fish scale powder is fully soaked in water, and then removed and drained; the scales are soaked in a 0.1-0.3M HCl solution equivalent to 5-15 times the weight of the scales at room temperature for 3-5 hours, and then rinsed with clean water; the washed fish scales are placed in a heating kettle with stirring, 5-10 times the weight of deionized water is added, and the scales are heated at 95-100°C with stirring for 6-8 hours to obtain fish scale glue; the obtained fish scale glue is filtered while hot to remove solid impurities; and the filtrate is cooled to room temperature to obtain crude collagen glue.