Preparation method of collagen tripeptide solution and application thereof

Abstract

The present application relates to a kind of preparation method of collagen tripeptide liquid and its application, belong to the technical field of bioactive peptide, preparation method includes the following steps: A1, fish skin raw material is pretreated, and fish skin homogenate is obtained;A2, fish skin homogenate pH value is adjusted to neutral, and 1-4% of the complex protease of fish skin quality is added to carry out enzymolysis, and collagen peptide crude extract is obtained;A3, enzyme is killed, and centrifugation is carried out, and supernatant is collected to carry out nanofiltration, and obtain filter component 1 and cut-off component 1;A4, cut-off component 1 is placed in hydrochloric acid solution, and hydrolysis is obtained hydrolysate;Hydrolysate is adjusted to neutral, then nanofiltration is carried out, and obtain filter component 2 and cut-off component 2;A5, after filter component 1 and filter component 2 are mixed, nanofiltration is carried out, and cut-off component 3 is collected, and collagen tripeptide liquid is obtained.The collagen tripeptide liquid prepared in the present application has high collagen tripeptide yield, and the purity is also effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a method for preparing collagen tripeptide liquid and its application. Background Technology

[0002] Collagen is the most abundant structural protein in animals, accounting for up to 30% of total protein content. It is abundant in livestock, poultry, and fish (accounting for approximately 10-25% of total protein), but it is not easily digested and absorbed by the human body after being ingested through a daily diet. Collagen peptides are small-molecule peptide products made by enzymatically hydrolyzing large collagen molecules. They have effects such as improving skin hydration, reducing wrinkles, and anti-aging. Furthermore, the lower the molecular weight of collagen peptides, the easier they are to be absorbed by the human body.

[0003] Collagen tripeptides are tripeptide products prepared from raw materials such as pig skin and fish skin using advanced bioengineering technology. Their average molecular weight is 280-600 Da. Due to their small molecular weight, collagen tripeptides are easily absorbed and utilized by the human body. However, existing methods for obtaining collagen tripeptides through enzymatic hydrolysis with various proteases result in low yields and low purity, severely restricting their in-depth development and application. Therefore, there is an urgent need to provide a collagen tripeptide preparation method that can solve the above problems. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing collagen tripeptide solution and its application. Fish skin is first enzymatically hydrolyzed with a complex protease, and then the hydrolysate is nanofiltered to separate collagen tripeptides with a molecular weight below 600-800 Da from collagen peptides with a molecular weight greater than 600-800 Da. Instead of directly hydrolyzing the enzymatic hydrolysate, the collagen peptides with a molecular weight greater than 600-800 Da are acid-hydrolyzed, effectively preventing the collagen tripeptides obtained from enzymatic hydrolysis from being acid-hydrolyzed into free amino acids, thereby improving the yield of collagen tripeptides and simultaneously increasing the acid hydrolysis efficiency.

[0005] The technical problem this invention aims to solve is that existing collagen tripeptides obtained through enzymatic hydrolysis with various proteases have low yields and low purity, which severely restricts the in-depth development and application of collagen tripeptides.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A method for preparing a collagen tripeptide solution includes the following steps:

[0008] A1. Pre-treat the fish skin raw material to obtain a fish skin homogenate;

[0009] Further, in step A1, the specific pretreatment process is as follows: after cleaning the fish skin, first soak it in sodium hydroxide solution, then soak it in hydrochloric acid solution, wash it until neutral, and then beat it to obtain a fish skin homogenate.

[0010] Furthermore, the ratio of fish skin, sodium hydroxide solution, and hydrochloric acid solution used is 1g:(10-20)mL:(10-15)mL.

[0011] Furthermore, the concentration of the sodium hydroxide solution is 0.05-0.2M.

[0012] Furthermore, the concentration of the hydrochloric acid solution is 0.05-0.2M.

[0013] Furthermore, the soaking time in sodium hydroxide solution is 15-25 minutes.

[0014] Furthermore, the soaking time in hydrochloric acid solution is 5-15 minutes.

[0015] A2. Adjust the pH of the fish skin homogenate to neutral, add 1-4% of the fish skin mass of compound protease, and enzymatically hydrolyze at 40-60℃ for 6-18 hours to obtain crude collagen peptide extract.

[0016] Furthermore, in step A2, the complex protease is a mixture of neutral protease, papain, and bromelain.

[0017] Furthermore, the mass ratio of neutral protease, papain and bromelain is (6-8):(4-5):(3-4).

[0018] A3. Inactivate the enzymes in the crude collagen peptide extract, centrifuge, collect the supernatant, and nanofilter the supernatant to obtain filtered component 1 and retained component 1.

[0019] Furthermore, in step A3, the nanofiltration membrane used for nanofiltration has a molecular weight cutoff of 600-800 Da.

[0020] A4. Place the retained component 1 in hydrochloric acid solution and hydrolyze it at 85-110℃ for 3-7 hours to obtain a hydrolysate; adjust the hydrolysate to neutral and then perform nanofiltration to obtain filtered component 2 and retained component 2. Discard retained component 2.

[0021] Furthermore, in step A4, the nanofiltration membrane used for nanofiltration has a molecular weight cutoff of 600-800 Da.

[0022] Furthermore, in step A4, the concentration of the hydrochloric acid solution is 1-1.5M.

[0023] A5. After mixing the filtered components 1 and 2, perform nanofiltration and collect the retained component 3 to obtain collagen tripeptide solution.

[0024] Furthermore, in step A5, the molecular weight cutoff of the nanofiltration membrane used for nanofiltration is 150 Da.

[0025] In the above process, the fish skin is first pretreated, followed by enzymatic hydrolysis with a complex protease. After hydrolysis, the collagen tripeptide with a molecular weight of less than 600-800 Da is separated from the collagen peptide with a molecular weight greater than 600-800 Da by nanofiltration. The collagen peptide with a molecular weight greater than 600-800 Da is then acid-hydrolyzed and nanofiltered to collect the collagen tripeptide with a molecular weight of less than 600-800 Da. The collagen tripeptides with a molecular weight of less than 600-800 Da obtained from the two nanofiltration processes are mixed and then desalted by nanofiltration to obtain high-purity collagen tripeptides. This invention involves enzymatic hydrolysis of fish skin homogenate with a complex protease. At this point, the collagen tripeptide yield is 36-39%. To improve the collagen tripeptide yield, the hydrolysate is first subjected to nanofiltration to separate collagen tripeptides with a molecular weight below 600-800 Da from collagen peptides with a molecular weight greater than 600-800 Da. The collagen peptides with a molecular weight greater than 600-800 Da are then acid-hydrolyzed instead of directly hydrolyzing the hydrolysate. This effectively prevents the collagen tripeptides obtained from enzymatic hydrolysis from being hydrolyzed into free amino acids, thereby increasing the collagen tripeptide yield and improving the acid hydrolysis efficiency. This invention, through a series of operations—enzymatic hydrolysis-nanofiltration-acid hydrolysis-nanofiltration-nanofiltration—significantly improves the purity of collagen tripeptides.

[0026] The present invention also provides the application of the collagen tripeptide liquid in cosmetics and functional foods.

[0027] The beneficial effects of this invention are:

[0028] (1) In the technical solution of the present invention, the collagen tripeptide has a high yield and high bioactivity, and its application in cosmetics and functional foods has significant effects and is loved by consumers.

[0029] (2) In the technical solution of the present invention, the fish skin is subjected to a combination of enzymatic hydrolysis with complex protease, nanofiltration, acid hydrolysis, nanofiltration and nanofiltration in sequence, which significantly improves the purity of collagen tripeptide. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] A method for preparing a collagen tripeptide solution includes the following steps:

[0033] A1. Remove the non-skin tissue from the tilapia skin, clean it thoroughly, soak it in 0.05M sodium hydroxide solution for 15 minutes, then soak it in 0.05M hydrochloric acid solution for 5 minutes, clean it until neutral, and then beat it to obtain a fish skin homogenate; the ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:10mL:10mL.

[0034] A2. Adjust the pH of the fish skin homogenate to neutral, add 1% of the fish skin mass of compound protease, and enzymatically hydrolyze at 40℃ for 18h to obtain crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the compound protease is 6:4:3.

[0035] A3. Treat the crude collagen peptide extract with enzyme inactivation at 100℃ for 10 min, centrifuge, collect the supernatant, and nanofilter the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 1 and retained component 1.

[0036] A4. Place the retained component 1 in a 1M hydrochloric acid solution (the volume ratio of retained component 1 to hydrochloric acid solution is 1:15) and hydrolyze it at 85℃ for 3 hours to obtain a hydrolysate. Adjust the hydrolysate to neutral and then pass it through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 2 and retained component 2. Discard retained component 2.

[0037] A5. After mixing the filtered components 1 and 2, the mixture is nanofiltered through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Component 3 is collected to obtain collagen tripeptide solution.

[0038] Example 2

[0039] A method for preparing a collagen tripeptide solution includes the following steps:

[0040] A1. Remove the non-skin tissue from the tilapia skin, clean it thoroughly, soak it in 0.1M sodium hydroxide solution for 20 minutes, then soak it in 0.1M hydrochloric acid solution for 10 minutes, clean it until neutral, and then homogenize it to obtain a fish skin paste; the ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:12mL:12mL.

[0041] A2. Adjust the pH of the fish skin homogenate to neutral, add 2% of the fish skin mass of compound protease, and enzymatically hydrolyze at 50℃ for 12 hours to obtain crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the compound protease is 7:4:3.2.

[0042] A3. Treat the crude collagen peptide extract with enzyme inactivation at 100℃ for 10 min, centrifuge, collect the supernatant, and nanofilter the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 1 and retained component 1.

[0043] A4. Place the retained component 1 in a 1.2M hydrochloric acid solution (the volume ratio of retained component 1 to hydrochloric acid solution is 1:15) and hydrolyze it at 90℃ for 5 hours to obtain a hydrolysate. Adjust the hydrolysate to neutral and then pass it through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 2 and retained component 2. Discard retained component 2.

[0044] A5. After mixing the filtered components 1 and 2, the mixture is nanofiltered through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Component 3 is collected to obtain collagen tripeptide solution.

[0045] Example 3

[0046] A method for preparing a collagen tripeptide solution includes the following steps:

[0047] A1. Remove the non-skin tissue from the tilapia skin, clean it thoroughly, soak it in 0.12M sodium hydroxide solution for 20 minutes, then soak it in 0.15M hydrochloric acid solution for 10 minutes, clean it until neutral, and then homogenize it to obtain a fish skin paste; the ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:15mL:12mL.

[0048] A2. Adjust the pH of the fish skin homogenate to neutral, add 2.5% of the fish skin mass of compound protease, and enzymatically hydrolyze at 50℃ for 14 hours to obtain crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the compound protease is 7:5:3.5.

[0049] A3. The crude collagen peptide extract was subjected to enzyme inactivation treatment at 100℃ for 12 min, centrifuged, and the supernatant was collected. The supernatant was then nanofiltered through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 1 and retained component 1.

[0050] A4. Place the retained component 1 in a 1.3M hydrochloric acid solution (the volume ratio of retained component 1 to hydrochloric acid solution is 1:15) and hydrolyze it at 95℃ for 5 hours to obtain a hydrolysate. Adjust the hydrolysate to neutral and then pass it through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 2 and retained component 2. Discard retained component 2.

[0051] A5. After mixing the filtered components 1 and 2, the mixture is nanofiltered through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Component 3 is collected to obtain collagen tripeptide solution.

[0052] Example 4

[0053] A method for preparing a collagen tripeptide solution includes the following steps:

[0054] A1. Remove the non-skin tissue from the tilapia skin, clean it thoroughly, soak it in 0.16M sodium hydroxide solution for 20 minutes, then soak it in 0.15M hydrochloric acid solution for 12 minutes, clean it until neutral, and then homogenize it to obtain a fish skin paste; the ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:17mL:14mL.

[0055] A2. Adjust the pH of the fish skin homogenate to neutral, add 3% of the fish skin mass of compound protease, and enzymatically hydrolyze at 55℃ for 15 hours to obtain crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the compound protease is 8:4:4.

[0056] A3. Treat the crude collagen peptide extract with enzyme inactivation at 100℃ for 15 min, centrifuge, collect the supernatant, and nanofilter the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 1 and retained component 1.

[0057] A4. Place the retained component 1 in a 1.5M hydrochloric acid solution (the volume ratio of retained component 1 to hydrochloric acid solution is 1:15) and hydrolyze it at 100℃ for 6 hours to obtain a hydrolysate. Adjust the hydrolysate to neutral and then pass it through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 2 and retained component 2. Discard retained component 2.

[0058] A5. After mixing the filtered components 1 and 2, the mixture is nanofiltered through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Component 3 is collected to obtain collagen tripeptide solution.

[0059] Example 5

[0060] A method for preparing a collagen tripeptide solution includes the following steps:

[0061] A1. Remove the non-skin tissue from the tilapia skin, clean it thoroughly, soak it in 0.2M sodium hydroxide solution for 25 minutes, then soak it in 0.2M hydrochloric acid solution for 15 minutes, clean it until neutral, and then homogenize it to obtain a fish skin paste; the ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:20mL:15mL.

[0062] A2. Adjust the pH of the fish skin homogenate to neutral, add 4% of the fish skin mass of compound protease, and enzymatically hydrolyze at 60℃ for 6 hours to obtain crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the compound protease is 8:5:4.

[0063] A3. Treat the crude collagen peptide extract with enzyme inactivation at 100℃ for 15 min, centrifuge, collect the supernatant, and nanofilter the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 1 and retained component 1.

[0064] A4. Place the retained component 1 in a 1.5M hydrochloric acid solution (the volume ratio of retained component 1 to hydrochloric acid solution is 1:15) and hydrolyze it at 110℃ for 7 hours to obtain a hydrolysate. Adjust the hydrolysate to neutral and then pass it through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 2 and retained component 2. Discard retained component 2.

[0065] A5. After mixing the filtered components 1 and 2, the mixture is nanofiltered through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Component 3 is collected to obtain collagen tripeptide solution.

[0066] Example 6

[0067] A method for preparing a collagen tripeptide solution includes the following steps:

[0068] A1. Remove the non-skin tissue from the tilapia skin, clean it thoroughly, soak it in 0.05M sodium hydroxide solution for 25 minutes, then soak it in 0.05M hydrochloric acid solution for 15 minutes, clean it until neutral, and then homogenize it to obtain a fish skin paste; the ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:20mL:10mL.

[0069] A2. Adjust the pH of the fish skin homogenate to neutral, add 4% of the fish skin mass of compound protease, and enzymatically hydrolyze at 60℃ for 6 hours to obtain crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the compound protease is 6:5:3.5.

[0070] A3. Treat the crude collagen peptide extract with enzyme inactivation at 100℃ for 15 min, centrifuge, collect the supernatant, and nanofilter the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 1 and retained component 1.

[0071] A4. Place the retained component 1 in a 1.2M hydrochloric acid solution (the volume ratio of retained component 1 to hydrochloric acid solution is 1:15) and hydrolyze it at 100℃ for 4 hours to obtain a hydrolysate. Adjust the hydrolysate to neutral and then pass it through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 2 and retained component 2. Discard retained component 2.

[0072] A5. After mixing the filtered components 1 and 2, the mixture is nanofiltered through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Component 3 is collected to obtain collagen tripeptide solution.

[0073] Example 7

[0074] A method for preparing a collagen tripeptide solution includes the following steps:

[0075] A1. Remove the non-skin tissue from the tilapia skin, clean it thoroughly, soak it in 0.1M sodium hydroxide solution for 20 minutes, then soak it in 0.1M hydrochloric acid solution for 5 minutes, clean it until neutral, and then homogenize it to obtain a fish skin paste; the ratio of fish skin, sodium hydroxide solution and hydrochloric acid solution is 1g:10mL:15mL.

[0076] A2. Adjust the pH of the fish skin homogenate to neutral, add 3% of the fish skin mass of compound protease, and enzymatically hydrolyze at 40℃ for 6 hours to obtain crude collagen peptide extract; the mass ratio of neutral protease, papain and bromelain in the compound protease is 8:4:3.

[0077] A3. Treat the crude collagen peptide extract with enzyme inactivation at 100℃ for 15 min, centrifuge, collect the supernatant, and nanofilter the supernatant through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 1 and retained component 1.

[0078] A4. Place the retained component 1 in a 1.5M hydrochloric acid solution (the volume ratio of retained component 1 to hydrochloric acid solution is 1:15) and hydrolyze it at 95℃ for 5 hours to obtain a hydrolysate. Adjust the hydrolysate to neutral and then pass it through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da to obtain filtered component 2 and retained component 2. Discard retained component 2.

[0079] A5. After mixing the filtered components 1 and 2, the mixture is nanofiltered through a nanofiltration membrane with a molecular weight cutoff of 150 Da. Component 3 is collected to obtain collagen tripeptide solution.

[0080] Comparative Example 1

[0081] Compared with Example 3, the complex protease in Comparative Example 1 was a neutral protease and papain, while the other steps and raw materials were the same as in Example 3.

[0082] Comparative Example 2

[0083] Compared with Example 3, the complex protease in Comparative Example 2 was a neutral protease and a bromelain, while the other steps and raw materials were the same as in Example 3.

[0084] Comparative Example 3

[0085] Compared with Example 3, the complex protease in Comparative Example 3 was papain and bromelain, while the other steps and raw materials were the same as in Example 3.

[0086] Comparative Example 4

[0087] Compared with Example 3, Comparative Example 4 only underwent enzymatic hydrolysis with a complex protease, and then was sequentially nanofiltered through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da and a nanofiltration membrane with a molecular weight cutoff of 150 Da. Other steps and raw materials were the same as in Example 3.

[0088] Comparative Example 5

[0089] Compared with Example 3, Comparative Example 5 was subjected to enzymatic hydrolysis with a complex protease followed by direct acid hydrolysis, and then sequentially passed through a nanofiltration membrane with a molecular weight cutoff of 600-800 Da and a nanofiltration membrane with a molecular weight cutoff of 150 Da. Other steps and raw materials were the same as in Example 3.

[0090] Performance testing

[0091] 1. The content of tripeptide in the collagen tripeptide products prepared in Examples 1-7 and Comparative Examples 1-5 was detected by HPLC-MS, and the results are shown in Table 1.

[0092] HPLC-MS detection method: ZORBAX SB-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 scan range m / z 300-1500; both precise mass number scan (Zoomscan) and secondary mass spectrometry (MS / MS) scans were data-dependent scans; dynamic exclusion count 1; dynamic exclusion time 0.5 min; secondary mass spectrometry collision energy 35%.

[0093] Table 1

[0094]

[0095] As can be seen from the data in Table 1, the collagen tripeptide prepared by this invention has high purity and the collagen tripeptide content can reach 90%.

[0096] 2. The filtration components 1, filtration components 2 and retention components 2 obtained in the preparation processes of Examples 1-7 and Comparative Examples 1-3 were mixed and their molecular weights were determined. The molecular weight of the enzymatic hydrolysate prepared in Comparative Example 4 was determined, and the molecular weight of the hydrolysate prepared in Comparative Example 5 after acid hydrolysis was determined. The proportion of products with molecular weights in the range of 180-600 Da after enzymatic and acid hydrolysis treatments was determined, i.e., the yield of collagen tripeptides. The molecular weights were determined according to the method in Appendix A of GB / T 22729-2008. The results are shown in Table 2.

[0097] Table 2

[0098]

[0099] As can be seen from the data in Table 2, the collagen tripeptides prepared by this invention have a high yield and a low content of free amino acids with a molecular weight less than 180 Da. Comparing the data from Example 3 and Comparative Examples 1, 2, and 3, it can be seen that there is a synergistic effect among neutral protease, papain, and bromelain; their combined enzymatic hydrolysis can increase the yield of collagen tripeptides. Comparing the data from Example 3 and Comparative Example 4, it can be seen that the yield of collagen tripeptides from the enzymatic hydrolysis product without acid hydrolysis is significantly reduced. Comparing the data from Example 3 and Comparative Example 5, it can be seen that direct acid hydrolysis after enzymatic hydrolysis leads to an increase in the content of free amino acids and a decrease in the yield of collagen tripeptides.

[0100] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0101] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method for preparing a collagen tripeptide solution, characterized in that: Includes the following steps: A1. Pre-treat the fish skin raw material to obtain a fish skin homogenate; A2. Adjust the pH of the fish skin homogenate to neutral, add 1-4% of the fish skin mass of compound protease, and enzymatically hydrolyze at 40-60℃ for 6-18 hours to obtain crude collagen peptide extract. In step A2, the complex 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). A3. Inactivate the enzymes in the crude collagen peptide extract, centrifuge, collect the supernatant, and perform nanofiltration on the supernatant to obtain filtration component 1 and retention component 1. In A3, the molecular weight cutoff of the nanofiltration membrane used for nanofiltration is 600-800 Da; A4. Place the retained component 1 in hydrochloric acid solution and hydrolyze it at 85-110℃ for 3-7 hours to obtain a hydrolysate; adjust the hydrolysate to neutral and then perform nanofiltration to obtain filtered component 2 and retained component 2. Discard retained component 2. In step A4, the molecular weight cutoff of the nanofiltration membrane used for nanofiltration is 600-800 Da; A5. After mixing the filtered components 1 and 2, nanofiltration is performed, and the retained component 3 is collected to obtain collagen tripeptide solution; In step A5, the nanofiltration membrane used for nanofiltration has a molecular weight cutoff of 150 Da.

2. The method for preparing a collagen tripeptide solution according to claim 1, characterized in that: In step A1, the pretreatment process is as follows: after cleaning the fish skin, first soak it in sodium hydroxide solution, then soak it in hydrochloric acid solution, wash it until neutral, and then 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 ratio of fish skin, sodium hydroxide solution, and hydrochloric acid solution used is 1g:(10-20)mL:(10-15)mL; the concentration of sodium hydroxide solution is 0.05-0.2M; and the concentration of hydrochloric acid solution is 0.05-0.2M.

4. The method for preparing a collagen tripeptide solution according to claim 2, characterized in that: The soaking time in sodium hydroxide solution is 15-25 minutes, and the soaking time in 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 A4, the concentration of the hydrochloric acid solution is 1-1.5M.

6. The application of the collagen tripeptide liquid prepared by the preparation method according to any one of claims 1-5 in cosmetics and functional foods.

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