Preparation method and application of collagen tripeptide liquid

Abstract

The invention relates to a preparation method and application of collagen tripeptide liquid, and belongs to the technical field of bioactive peptides, the preparation method comprises the following steps: A1, pretreating fish skin raw materials to obtain fish skin homogenate; a2, the pH value of the fish skin homogenate is adjusted to be neutral, compound protease accounting for 1-4% of the mass of the fish skin is added for enzymolysis, and a collagen peptide crude extracting solution is obtained; a3, performing enzyme deactivation and centrifugation, collecting supernate, and performing nanofiltration to obtain a filtered component 1 and an intercepted component 1; a4, putting the interception component 1 into a hydrochloric acid solution, and hydrolyzing to obtain hydrolysate; adjusting the hydrolysate to be neutral, and then carrying out nanofiltration to obtain a filtered component 2 and an intercepted component 2; and A5, mixing the filtered component 1 and the filtered component 2, carrying out nanofiltration, and collecting an intercepted component 3 to obtain a collagen tripeptide solution. The collagen tripeptide in the prepared collagen tripeptide liquid is high in yield, and the purity is also effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive peptides, and in particular relates to a preparation method of a collagen tripeptide liquid and an application thereof. Background Art

[0002] Collagen is the most abundant structural protein in animals, accounting for 30% of the total protein content. It is abundant in livestock, poultry and fish (accounting for about 10-25% of the total protein), but it is not easily digested and absorbed by the human body after daily dietary intake. Collagen peptides are small molecule peptide products made by enzymatic hydrolysis of macromolecular collagen. They have the effects of increasing skin moisture, reducing wrinkles, and anti-aging. The lower the molecular weight of collagen peptides, the easier it is to be absorbed by the human body.

[0003] Collagen tripeptide is a tripeptide product prepared from pig skin, fish skin, etc. using advanced bioengineering technology. Its average molecular weight is 280-600Da. Since the molecular weight of collagen tripeptide is very small, it is easily absorbed and utilized by the human body. However, the existing collagen tripeptide obtained by enzymatic hydrolysis of multiple proteases has a low yield and low purity, which seriously restricts the in-depth development and application of collagen tripeptide. Therefore, it is urgent to provide a method for preparing collagen tripeptide that can solve the above problems. Summary of the invention

[0004] The invention aims to provide a preparation method and application of a collagen tripeptide liquid. Fish skin is firstly enzymatically hydrolyzed with a composite protease, and then the enzymatic hydrolyzate is nanofiltered to separate collagen tripeptides with a molecular weight of less than 600-800Da and collagen peptides with a molecular weight greater than 600-800Da. The collagen peptides with a molecular weight greater than 600-800Da are acid-hydrolyzed instead of directly acid-hydrolyzing the enzymatic hydrolyzate, so that the collagen tripeptides obtained by the enzymatic hydrolysis are effectively avoided from being acid-hydrolyzed into free amino acids, thereby improving the yield of the collagen tripeptides and the acid hydrolysis efficiency.

[0005] The technical problem to be solved by the present invention is that the existing collagen tripeptide obtained by enzymatic hydrolysis with a variety of proteases has a low yield and low purity, which seriously restricts the in-depth development and application of collagen tripeptide.

[0006] The purpose of the present invention can be achieved through the following technical solutions: A method for preparing a collagen tripeptide solution comprises the following steps: A1, the fish skin raw material is pre-treated to obtain fish skin homogenate; Furthermore, in step A1, the specific process of pretreatment is: after cleaning the fish skin, first soak it in a sodium hydroxide solution, then soak it in a hydrochloric acid solution, wash it until it is neutral, and beat it to obtain a fish skin homogenate.

[0007] Furthermore, the usage ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1 g: (10-20) mL: (10-15) mL.

[0008] Furthermore, the concentration of the sodium hydroxide solution is 0.05-0.2M.

[0009] Furthermore, the concentration of the hydrochloric acid solution is 0.05-0.2M.

[0010] Furthermore, the soaking time in the sodium hydroxide solution is 15-25 minutes.

[0011] Furthermore, the soaking time in the hydrochloric acid solution is 5-15 minutes.

[0012] A2, the fish skin homogenate pH value is adjusted to neutral, the fish skin mass 1-4% composite protease is added, and enzymolysis is carried out at 40-60 ℃ for 6-18h to obtain a crude extract of collagen peptides; Furthermore, in step A2, the composite protease is a mixture of neutral protease, papain and bromelain.

[0013] Furthermore, the mass ratio of neutral protease, papain and bromelain is (6-8):(4-5):(3-4).

[0014] A3, inactivating enzymes in the crude collagen peptide extract, centrifuging, collecting the supernatant, and nanofiltering the supernatant to obtain a filtered component 1 and a retained component 1; Furthermore, in step A3, the molecular weight cut-off of the nanofiltration membrane used for nanofiltration is 600-800Da.

[0015] A4, placing the retained component 1 in a hydrochloric acid solution, hydrolyzing it at 85-110°C for 3-7h to obtain a hydrolyzate; adjusting the hydrolyzate to neutrality, and then performing nanofiltration to obtain a filtered component 2 and a retained component 2, and discarding the retained component 2; Furthermore, in step A4, the molecular weight cut-off of the nanofiltration membrane used for nanofiltration is 600-800Da.

[0016] Furthermore, in step A4, the concentration of the hydrochloric acid solution is 1-1.5M.

[0017] A5. Mix the filtered component 1 and the filtered component 2 and perform nanofiltration, collect the retained component 3, and obtain the collagen tripeptide solution.

[0018] Furthermore, in step A5, the molecular weight cutoff of the nanofiltration membrane used for nanofiltration is 150Da.

[0019] In the above-mentioned operation process, first fish skin is pre-treated, and composite protease enzymolysis is carried out after the pre-treatment, and after the enzymolysis, it is first filtered through nanofiltration membrane, and the collagen tripeptide with a molecular weight below 600-800Da is separated from the collagen peptide with a molecular weight greater than 600-800Da, and the collagen peptide with a molecular weight greater than 600-800Da is acid-hydrolyzed again, and nanofiltration is carried out after the acidolysis to collect the collagen tripeptide with a molecular weight below 600-800Da, and the collagen tripeptide with a molecular weight below 600-800Da obtained by twice of nanofiltration is mixed, and then desalted through nanofiltration to obtain high-purity collagen tripeptide. The present invention performs composite protease enzymolysis on fish skin homogenate, and the collagen tripeptide yield is 36-39% at this time. In order to improve the collagen tripeptide yield, the present invention first performs nanofiltration on the enzymolysis solution, separates the collagen tripeptide with a molecular weight below 600-800Da and the collagen peptide with a molecular weight greater than 600-800Da, and performs acid hydrolysis on the collagen peptide with a molecular weight greater than 600-800Da, instead of directly performing acid hydrolysis on the enzymolysis solution, effectively avoiding the collagen tripeptide obtained by enzymolysis from being acid-hydrolyzed into free amino acids, thereby improving the collagen tripeptide yield and the acid hydrolysis efficiency. The present invention significantly improves the purity of the collagen tripeptide through a series of operations of enzymolysis-nanofiltration-acid hydrolysis-nanofiltration-nanofiltration.

[0020] The invention also provides application of the collagen tripeptide liquid in cosmetics and functional foods.

[0021] Beneficial effects of the present invention: (1) In the technical solution of the present invention, the collagen tripeptide has a high yield and high biological activity, and has significant effects when applied in cosmetics and functional foods, and is deeply loved by consumers.

[0022] (2) In the technical solution of the present invention, the fish skin is subjected to a composite protease hydrolysis-nanofiltration-acid hydrolysis-nanofiltration-nanofiltration operation in sequence, which significantly improves the purity of the collagen tripeptide. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1 A method for preparing a collagen tripeptide solution comprises the following steps: A1, tilapia fish skin is removed to non-fish skin tissue, after cleaning, first put into 0.05M sodium hydroxide solution and soak for 15min, then put into 0.05M hydrochloric acid solution and soak for 5min, clean to neutrality, beat an oar, obtain fish skin homogenate; the amount ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:10mL:10mL; A2, the pH value of the fish skin homogenate was adjusted to neutral, and a composite protease of 1% by weight of the fish skin was added, and enzymolysis was performed at 40°C for 18 hours to obtain a crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the composite protease was 6:4:3; A3, treating the collagen peptide crude extract at 100°C for 10 minutes by enzyme inactivation, centrifuging, collecting the supernatant, and filtering the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 1 and a retained component 1; A4, placing the retained component 1 in a 1M hydrochloric acid solution (the volume ratio of the retained component 1 to the hydrochloric acid solution is 1:15), and hydrolyzing at 85°C for 3 hours to obtain a hydrolyzate; adjusting the hydrolyzate to neutrality, and then nanofiltering through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 2 and a retained component 2, and discarding the retained component 2; A5. Mix the filtered component 1 and the filtered component 2, and filter them through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Collect the retained component 3 to obtain a collagen tripeptide solution.

[0025] Example 2 A method for preparing a collagen tripeptide solution comprises the following steps: A1, tilapia fish skin is removed to non-fish skin tissue, after cleaning, first put into 0.1M sodium hydroxide solution and soak for 20min, then put into 0.1M hydrochloric acid solution and soak for 10min, clean to neutrality, beat an oar, obtain fish skin homogenate; the amount ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:12mL:12mL; A2, the pH value of the fish skin homogenate was adjusted to neutral, and a composite protease of 2% by weight of the fish skin was added, and enzymolysis was performed at 50°C for 12 hours to obtain a crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the composite protease was 7:4:3.2; A3, treating the collagen peptide crude extract at 100°C for 10 minutes by enzyme inactivation, centrifuging, collecting the supernatant, and filtering the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 1 and a retained component 1; A4, placing the retained component 1 in a 1.2M hydrochloric acid solution (the volume ratio of the retained component 1 to the hydrochloric acid solution is 1:15), and hydrolyzing at 90°C for 5 hours to obtain a hydrolyzate; adjusting the hydrolyzate to neutrality, and then nanofiltering through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 2 and a retained component 2, and discarding the retained component 2; A5. Mix the filtered component 1 and the filtered component 2, and filter them through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Collect the retained component 3 to obtain a collagen tripeptide solution.

[0026] Example 3 A method for preparing a collagen tripeptide solution comprises the following steps: A1, tilapia fish skin is removed to non-fish skin tissue, after cleaning, first put into 0.12M sodium hydroxide solution and soak for 20min, then put into 0.15M hydrochloric acid solution and soak for 10min, clean to neutrality, beat an oar, obtain fish skin homogenate; the amount ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:15mL:12mL; A2, the pH value of the fish skin homogenate was adjusted to neutral, and 2.5% of the weight of the fish skin composite protease was added, and enzymolysis was performed at 50°C for 14h to obtain a crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the composite protease was 7:5:3.5; A3, treating the collagen peptide crude extract at 100°C for 12 minutes by enzyme inactivation, centrifuging, collecting the supernatant, and filtering the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 1 and a retained component 1; A4, placing the retained component 1 in a 1.3M hydrochloric acid solution (the volume ratio of the retained component 1 to the hydrochloric acid solution is 1:15), and hydrolyzing at 95°C for 5 hours to obtain a hydrolyzate; adjusting the hydrolyzate to neutrality, and then nanofiltering through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 2 and a retained component 2, and discarding the retained component 2; A5. Mix the filtered component 1 and the filtered component 2, and filter them through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Collect the retained component 3 to obtain a collagen tripeptide solution.

[0027] Example 4 A method for preparing a collagen tripeptide solution comprises the following steps: A1, tilapia fish skin is removed to non-fish skin tissue, after cleaning, first put into 0.16M sodium hydroxide solution and soak for 20min, then put into 0.15M hydrochloric acid solution and soak for 12min, clean to neutrality, beat an oar, obtain fish skin homogenate; the amount ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:17mL:14mL; A2, the pH value of the fish skin homogenate was adjusted to neutral, and a composite protease of 3% by weight of the fish skin was added, and enzymolysis was performed at 55°C for 15h to obtain a crude collagen peptide extract; the mass ratio of the neutral protease, papain and bromelain in the composite protease was 8:4:4; A3, inactivating the enzyme in the crude collagen peptide extract at 100°C for 15 minutes, centrifuging, collecting the supernatant, and filtering the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 1 and a retained component 1; A4, placing the retained component 1 in a 1.5M hydrochloric acid solution (the volume ratio of the retained component 1 to the hydrochloric acid solution is 1:15), and hydrolyzing at 100°C for 6 hours to obtain a hydrolyzate; adjusting the hydrolyzate to neutrality, and then nanofiltering through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 2 and a retained component 2, and discarding the retained component 2; A5. Mix the filtered component 1 and the filtered component 2, and filter them through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Collect the retained component 3 to obtain a collagen tripeptide solution.

[0028] Example 5 A method for preparing a collagen tripeptide solution comprises the following steps: A1, tilapia fish skin is removed to non-fish skin tissue, after cleaning, first put into 0.2M sodium hydroxide solution and soak for 25min, then put into 0.2M hydrochloric acid solution and soak for 15min, clean to neutrality, beat an oar, obtain fish skin homogenate; the amount ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:20mL:15mL; A2, the pH value of the fish skin homogenate was adjusted to neutral, and a composite protease of 4% by weight of the fish skin was added, and enzymolysis was performed at 60°C for 6 hours to obtain a crude collagen peptide extract; the mass ratio of the neutral protease, papain and bromelain in the composite protease was 8:5:4; A3, inactivating the enzyme in the crude collagen peptide extract at 100°C for 15 minutes, centrifuging, collecting the supernatant, and filtering the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 1 and a retained component 1; A4, placing the retained component 1 in a 1.5M hydrochloric acid solution (the volume ratio of the retained component 1 to the hydrochloric acid solution is 1:15), and hydrolyzing at 110°C for 7 hours to obtain a hydrolyzate; adjusting the hydrolyzate to neutrality, and then nanofiltering through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 2 and a retained component 2, and discarding the retained component 2; A5. Mix the filtered component 1 and the filtered component 2, and filter them through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Collect the retained component 3 to obtain a collagen tripeptide solution.

[0029] Example 6 A method for preparing a collagen tripeptide solution comprises the following steps: A1, tilapia fish skin is removed to non-fish skin tissue, after cleaning, first put into 0.05M sodium hydroxide solution and soak for 25min, then put into 0.05M hydrochloric acid solution and soak for 15min, clean to neutrality, beat an oar, obtain fish skin homogenate; the amount ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:20mL:10mL; A2, the pH value of the fish skin homogenate was adjusted to neutral, 4% of the fish skin mass of the composite protease was added, and enzymolysis was carried out at 60 ° C for 6 h to obtain a crude collagen peptide extract; the mass ratio of the neutral protease, papain and bromelain in the composite protease was 6:5:3.5; A3, inactivating the enzyme in the crude collagen peptide extract at 100°C for 15 minutes, centrifuging, collecting the supernatant, and filtering the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 1 and a retained component 1; A4, placing the retained component 1 in a 1.2M hydrochloric acid solution (the volume ratio of the retained component 1 to the hydrochloric acid solution is 1:15), and hydrolyzing at 100°C for 4 hours to obtain a hydrolyzate; adjusting the hydrolyzate to neutrality, and then nanofiltration through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 2 and a retained component 2, and discarding the retained component 2; A5. Mix the filtered component 1 and the filtered component 2, and filter them through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Collect the retained component 3 to obtain a collagen tripeptide solution.

[0030] Example 7 A method for preparing a collagen tripeptide solution comprises the following steps: A1, tilapia fish skin is removed to non-fish skin tissue, after cleaning, first put into 0.1M sodium hydroxide solution and soak for 20min, then put into 0.1M hydrochloric acid solution and soak for 5min, clean to neutrality, beat an oar, obtain fish skin homogenate; the amount ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:10mL:15mL; A2, the pH value of the fish skin homogenate was adjusted to neutral, and a composite protease of 3% by weight of the fish skin was added, and enzymolysis was performed at 40°C for 6 hours to obtain a crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the composite protease was 8:4:3; A3, inactivating the enzyme in the crude collagen peptide extract at 100°C for 15 minutes, centrifuging, collecting the supernatant, and filtering the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 1 and a retained component 1; A4, placing the retained component 1 in a 1.5M hydrochloric acid solution (the volume ratio of the retained component 1 to the hydrochloric acid solution is 1:15), and hydrolyzing at 95°C for 5 hours to obtain a hydrolyzate; adjusting the hydrolyzate to neutrality, and then nanofiltering through a nanofiltration membrane with a molecular weight cutoff of 600-800Da to obtain a filtered component 2 and a retained component 2, and discarding the retained component 2; A5. Mix the filtered component 1 and the filtered component 2, and filter them through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Collect the retained component 3 to obtain a collagen tripeptide solution.

[0031] Comparative Example 1 Compared with Example 3, the composite protease in Comparative Example 1 is neutral protease and papain, and the other steps and raw materials are the same as those in Example 3.

[0032] Comparative Example 2 Compared with Example 3, the composite protease in Comparative Example 2 is neutral protease and bromelain, and the other steps and raw materials are the same as those in Example 3.

[0033] Comparative Example 3 Compared with Example 3, the composite protease in Comparative Example 3 is papain and bromelain, and the other steps and raw materials are the same as those in Example 3.

[0034] Comparative Example 4 Compared with Example 3, Comparative Example 4 only performs composite protease hydrolysis, and then sequentially performs nanofiltration through a nanofiltration membrane with a molecular weight cutoff of 600-800Da and a nanofiltration membrane with a molecular weight cutoff of 150Da. The other steps and raw materials are synchronized with Example 3.

[0035] Comparative Example 5 Compared with Example 3, in Comparative Example 5, acid hydrolysis is directly performed after enzymatic hydrolysis with the composite protease, and then nanofiltration is performed successively through a nanofiltration membrane with a molecular weight cutoff of 600-800Da and a nanofiltration membrane with a molecular weight cutoff of 150Da. The other steps and raw materials are synchronized with Example 3.

[0036] Performance Testing 1. HPLC-MS was used to detect the contents of tripeptides in the collagen tripeptide products obtained in Examples 1-7 and Comparative Examples 1-5. The results are shown in Table 1. HPLC-MS detection method: ZORBAXSB-C18 column (2.1 mm × 150 mm, 5 μm); mobile phase A-water (0.1% TFA), B-acetonitrile (0.1% TFA); gradient elution, 0-7 min, 5%-20% B; 7-50 min, 20%-32% B; 50-90 min, 32%-72% B; injection volume 50 μL; flow rate 0.2 mL·min -1. Spray voltage 4.5 kV; capillary temperature 300 °C; nitrogen (N2) 253 kPa; positive ion mode, primary mass spectrometry scanning range m / z 300-1500, accurate mass number scanning (Zoomscan) and secondary mass spectrometry (MS / MS) scanning are data dependent scanning (Data Dependent Scan); dynamic exclusion times 1; dynamic exclusion time 0.5 min; secondary mass spectrometry collision energy 35%.

[0037] Table 1

[0038] It can be seen from the data in Table 1 that the collagen tripeptide prepared by the present invention has high purity and the collagen tripeptide content can reach 90%.

[0039] 2. The filtered component 1, the filtered component 2 and the retained component 2 obtained in the preparation process of Examples 1-7 and Comparative Examples 1-3 were mixed and the molecular weight was measured. The molecular weight of the enzymatic hydrolyzate prepared in Comparative Example 4 was measured, and the molecular weight of the hydrolyzate after acid hydrolysis prepared in Comparative Example 5 was measured. The proportion of products with a molecular weight in the range of 180-600Da after enzymatic hydrolysis and acid hydrolysis was determined, that is, the yield of collagen tripeptide. The molecular weight was determined by referring to the method in Appendix A of GB / T 22729-2008. The results are shown in Table 2.

[0040] Table 2

[0041] As can be seen from the data in Table 2, the collagen tripeptide yield prepared by the present invention is high, and the free amino acid content of the molecular weight less than 180Da is low. It can be seen from the data of Comparative Example 3 and Comparative Examples 1, 2, and 3 that there is a synergistic effect between neutral protease, papain, and bromelain, and the three of them can improve the collagen tripeptide yield by co-enzymatic hydrolysis. It can be seen from the data of Comparative Example 3 and Comparative Example 4 that the yield of collagen tripeptide of the enzymatic hydrolysis product without acid hydrolysis is significantly reduced. It can be seen from the data of Comparative Example 3 and Comparative Example 5 that due to the direct acid hydrolysis after enzymatic hydrolysis, the free amino acid content is increased and the collagen tripeptide yield is reduced.

[0042] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0043] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for preparing a collagen tripeptide solution, characterized in that: The following steps are involved: A1, the fish skin raw material is pre-treated to obtain fish skin homogenate; A2, the fish skin homogenate pH value is adjusted to neutral, the fish skin mass 1-4% composite protease is added, and enzymolysis is carried out at 40-60 ℃ for 6-18h to obtain a crude extract of collagen peptides; A3, inactivating enzymes in the crude collagen peptide extract, centrifuging, collecting the supernatant, and nanofiltering the supernatant to obtain a filtered component 1 and a retained component 1; A4, placing the retained component 1 in a hydrochloric acid solution, hydrolyzing it at 85-110°C for 3-7h to obtain a hydrolyzate; adjusting the hydrolyzate to neutrality, and then performing nanofiltration to obtain a filtered component 2 and a retained component 2, and discarding the retained component 2; A5. Mix the filtered component 1 and the filtered component 2 and perform nanofiltration, collect the retained component 3, and obtain the collagen tripeptide solution.

2. The method for preparing a collagen tripeptide solution according to claim 1, characterized in that: In the step A1, the specific process of pretreatment is: after cleaning the fish skin, first soak it in a sodium hydroxide solution, then soak it in a hydrochloric acid solution, wash it until it is neutral, and beat it to obtain a fish skin homogenate.

3. The method for preparing a collagen tripeptide solution according to claim 2, characterized in that: The usage ratio of the fish skin, the sodium hydroxide solution and the hydrochloric acid solution is 1g:(10-20)mL:(10-15)mL.

4. The method for preparing a collagen tripeptide solution according to claim 2, characterized in that: The soaking time in the sodium hydroxide solution is 15-25 minutes, and the soaking time in the hydrochloric acid solution is 5-15 minutes.

5. The method for preparing a collagen tripeptide solution according to claim 1, characterized in that: In step A2, the composite protease is a mixture of neutral protease, papain and bromelain; the mass ratio of the neutral protease, papain and bromelain is (6-8):(4-5):(3-4).

6. The method for preparing a collagen tripeptide solution according to claim 1, characterized in that: In the A3, the molecular weight cut-off of the nanofiltration membrane used for nanofiltration is 600-800Da.

7. The method for preparing a collagen tripeptide solution according to claim 1, characterized in that: In step A4, the concentration of the hydrochloric acid solution is 1-1.5M.

8. The method for preparing a collagen tripeptide solution according to claim 1, characterized in that: In the step A4, the molecular weight cut-off of the nanofiltration membrane used for nanofiltration is 600-800Da.

9. The method for preparing a collagen tripeptide solution according to claim 1, characterized in that: In the step A5, the molecular weight cut-off of the nanofiltration membrane used for nanofiltration is 150 Da.

10. Use of the collagen tripeptide liquid prepared by the preparation method according to any one of claims 1 to 9 in cosmetics and functional foods.

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