A collagen tripeptide, and a preparation method and application thereof

Abstract

The application provides a collagen tripeptide and a preparation method and application thereof, and belongs to the technical field of active peptides. The application provides a preparation method of the collagen tripeptide. Tilapia scales are subjected to enzymolysis treatment with a high-temperature-resistant protease, and then subjected to enzymolysis treatment with alkaline protease and neutral protease in sequence. The enzymolysis liquid is filtered with a ceramic membrane and an organic filter membrane in sequence, and is subjected to interception with a 400 Da nanofiltration membrane. The permeate liquid contains active peptides mainly composed of the collagen tripeptide. The permeate liquid is concentrated and dried, and the collagen tripeptide powder is detected. The protein content is 96.2%, the GPH content is 3.8%, and the CTP content is 35.09%. The collagen tripeptide powder has good DPPH scavenging activity. The collagen tripeptide powder has obvious ROS scavenging efficacy, beta-galactosidase activity inhibition efficacy and telomerase activity enhancement efficacy on a zebrafish aging model induced by hydrogen peroxide, and has obvious anti-aging effect.

Description

Technical Field

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

[0002] With the increasing global trend of population aging, anti-aging has become a research hotspot in the fields of biomedicine, nutritional science, and cosmetics. The aging process involves multiple biological mechanisms, including oxidative stress, inflammatory response, apoptosis, and matrix degradation. Collagen, as the main structural protein of tissues such as skin, bone, and cartilage, plays a crucial role in maintaining tissue structure and function. However, with age, the body's ability to synthesize collagen gradually declines, leading to aging phenomena such as loose skin, wrinkle formation, bone fragility, and joint dysfunction.

[0003] Currently, there are numerous anti-aging products on the market based on collagen, with collagen peptides being the most common. Collagen peptides are small-molecule peptides obtained through enzymatic hydrolysis of collagen, making them easily absorbed by the body. They possess multiple biological activities, including promoting collagen synthesis, anti-oxidation, and moisturizing. However, traditional collagen peptide products are mostly mixed peptides with a wide molecular weight distribution and unclear active ingredients, affecting the stability and reliability of their anti-aging effects.

[0004] Against this backdrop, collagen tripeptides, as oligopeptides with a specific molecular weight (approximately 300–500 Da), have gradually become a focus of anti-aging research due to their structural stability, ease of absorption, and well-defined activity. Existing studies have shown that collagen tripeptides possess significant antioxidant, anti-inflammatory, and extracellular matrix synthesis-promoting abilities, effectively slowing skin aging and improving joint health. However, existing methods for preparing collagen tripeptides suffer from low yields, low purity, and complex processes, limiting their widespread application in anti-aging products. Summary of the Invention

[0005] This invention provides a collagen tripeptide, its preparation method, and its application. The collagen tripeptide has a high yield and a simple process, and the prepared collagen tripeptide has high purity.

[0006] The present invention provides a method for preparing collagen tripeptide, comprising the following steps: (1) mixing an aqueous solution of a thermostable protease with tilapia scales for high-temperature enzymatic hydrolysis pretreatment to obtain a pretreatment solution; the temperature of the high-temperature enzymatic hydrolysis pretreatment is 80-85℃;

[0007] (2) The pretreatment solution described in step (1) was enzymatically hydrolyzed and then inactivated by alkaline protease and neutral protease respectively to obtain the enzymatic hydrolysate.

[0008] (3) The enzymatic hydrolysate is filtered sequentially using a ceramic membrane and an organic filter membrane to obtain the filtrate;

[0009] (4) Use a nanofiltration membrane with a molecular weight cutoff of 400 Da to retain the permeate, which contains collagen tripeptide.

[0010] In one specific embodiment of the present invention, the pH value of the high-temperature enzymatic hydrolysis pretreatment in step (1) is 8.8 to 9.0, and the high-temperature enzymatic hydrolysis pretreatment time is 1 to 1.5 h.

[0011] In one specific embodiment of the present invention, the mass ratio of the thermoresistant protease and tilapia scales in step (1) is (0.8-1) kg: 1000 kg.

[0012] In one specific embodiment of the present invention, the enzymatic hydrolysis pH of the alkaline protease in step (2) is 8.8 to 9.0, and the enzymatic hydrolysis time is 3h to 3.5kg.

[0013] In one specific embodiment of the present invention, the enzymatic hydrolysis time of the neutral protease in step (2) is 1 h to 1.5 kg.

[0014] In one specific embodiment of the present invention, the pore size of the ceramic membrane in step (3) is 0.15 to 0.2 μm, and the processing pressure is 1.2 to 1.5 MPa;

[0015] The organic filter membrane has a pore size of 8–10 nm and a processing pressure of 2.0–2.3 MPa.

[0016] In one specific embodiment of the present invention, after collecting the permeate in step (4), the process further includes concentration and drying to obtain collagen tripeptide powder.

[0017] The present invention also provides a collagen tripeptide prepared using the above-described preparation method.

[0018] This invention also provides the application of the above-mentioned collagen tripeptide in the preparation of anti-aging products.

[0019] The present invention also provides an anti-aging product, the active ingredient of which includes the above-mentioned collagen tripeptide, and also includes excipients.

[0020] Beneficial effects: This invention provides a method for preparing collagen tripeptides. Compared with the prior art, tilapia scales do not require pretreatment. Instead, tilapia scales are directly enzymatically hydrolyzed with a thermostable protease, followed by enzymatic hydrolysis with alkaline protease and neutral protease in sequence. The hydrolysate is then filtered through a ceramic membrane and an organic filter membrane in sequence, and then retained using a nanofiltration membrane. The permeate with a molecular weight of less than 400 Da is collected. The permeate contains active peptides mainly composed of collagen tripeptides.

[0021] The present invention concentrates and dries the permeate, and then uses collagen tripeptide powder for testing. The protein content is as high as 96.2% and the ash content is 1.1%. Moreover, the collagen tripeptide powder contains 3.8% GPH (Gly-Pro-Hyp) and 35.09% CTP (collagen tripeptide).

[0022] This invention evaluates the in vitro antioxidant activity of the prepared collagen tripeptide powder, and the IC50 of collagen tripeptide in scavenging DPPH is [not specified]. 50 The value is close to that of vitamin C, indicating good DPPH scavenging activity. Furthermore, zebrafish tests were conducted on the collagen tripeptide powder. The MTC values ​​of the collagen tripeptide powder in the model zebrafish were 2000 and 250 μg / mL, respectively, suggesting that the collagen tripeptide powder has significant ROS scavenging effects, β-galactosidase activity inhibition effects, and telomerase activity enhancement effects in the hydrogen peroxide-induced zebrafish aging model, demonstrating a significant anti-aging effect to meet the current demand for high-quality active peptides. Attached Figure Description

[0023] Figure 1 Here is the liquid chromatogram of GPH standard;

[0024] Figure 2 The liquid chromatogram of collagen tripeptide GPH;

[0025] Figure 3 The liquid chromatogram of CTP standard;

[0026] Figure 4 The liquid chromatogram for CTP detection of collagen tripeptide;

[0027] Figure 5 This is a typical image of zebrafish β-galactosidase staining intensity after sample treatment. In the image, blue represents the staining intensity of β-galactosidase, and the darker the color, the stronger the activity. Detailed Implementation

[0028] The present invention provides a method for preparing collagen tripeptide, comprising the following steps: (1) mixing an aqueous solution of a thermostable protease with tilapia scales for high-temperature enzymatic hydrolysis pretreatment to obtain a pretreatment solution; the temperature of the high-temperature enzymatic hydrolysis pretreatment is 80-85℃;

[0029] (2) The pretreatment solution described in step (1) was enzymatically hydrolyzed and then inactivated by alkaline protease and neutral protease respectively to obtain the enzymatic hydrolysate.

[0030] (3) The enzymatic hydrolysate is filtered sequentially using a ceramic membrane and an organic filter membrane to obtain the filtrate;

[0031] (4) Use a nanofiltration membrane with a molecular weight cutoff of 400 Da to retain the permeate, which contains collagen tripeptide.

[0032] In preparing the aqueous solution of the thermostable protease described in this invention, for example in one embodiment, 12m³ of solution is added to the enzymatic hydrolysis vessel. 3 Deionized water was used, and the pH was adjusted to 8.8–9.0 using 1M sodium hydroxide. The mixture was then heated to 80–85°C, and 1 kg of thermostable protease (DuPont Danisco PHT) was added. The protease was thoroughly dissolved by stirring to obtain an aqueous solution of the thermostable protease. The stirring speed can be 60 rpm. The thermostable protease aqueous solution was mixed with tilapia scales. In one embodiment, 1000 kg of tilapia scales were added to the thermostable protease aqueous solution, and the mixture underwent high-temperature enzymatic hydrolysis pretreatment for 1 hour.

[0033] The present invention performs enzymatic hydrolysis on the pretreatment solution. The enzymatic hydrolysis includes sequential enzymatic hydrolysis with alkaline protease and neutral protease. For example, in one embodiment, 2 kg of alkaline protein (Xiasheng Industrial Group Co., Ltd., FDY-2241) is directly added to the aforementioned enzymatic hydrolysis tank, stirred and dissolved thoroughly, and after measuring the pH, the pH is adjusted to 8.8-9.0 with 1M sodium hydroxide. After stirring and hydrolyzing thoroughly for 3 hours, 2 kg of neutral protease (Xiasheng Industrial Group Co., Ltd., FDG-223) is added and hydrolyzed for 1 hour. Then, the temperature is raised to 100°C for enzyme inactivation treatment.

[0034] This invention provides a filtration process for the enzyme hydrolysate after enzyme inactivation, comprising sequential ceramic membrane filtration and organic membrane filtration. Specifically, in one embodiment, the enzyme hydrolysate is filtered through a ceramic membrane with a pore size of 0.2 μm at a pressure of 1.2 MPa and a stable flow rate of 2000 L / h. The ceramic membrane effectively filters out large fat molecules, proteins, and other impurities, significantly improving product quality. The filtrate from the ceramic membrane filtration is then filtered through an organic membrane with a pore size of 10 nm at a pressure of 2.3 MPa and a stable flow rate of 2800 L / h. The ceramic membrane filtration method of this invention effectively removes large molecular impurities and proteins that have not undergone enzymatic hydrolysis.

[0035] This invention involves retaining the filtrate after filtration by an organic filtration membrane. In one specific embodiment, it includes using a nanofiltration membrane with a molecular weight cutoff of 400 Da to retain the filtrate and collecting the permeate with a molecular weight of less than 400 Da. The permeate contains the main active peptides, primarily collagen tripeptides.

[0036] The present invention concentrates the permeate, for example, in one specific embodiment using a high-pressure reverse osmosis membrane to increase the liquid concentration to 32% to 36%, and then performs spray drying. In one embodiment, the spray drying conditions can be an inlet air temperature of 135°C and an outlet air temperature of 75°C, ultimately yielding collagen tripeptide powder.

[0037] The present invention also provides a collagen tripeptide prepared using the above-described preparation method.

[0038] This invention also includes physicochemical testing of the collagen tripeptide, including determining the protein content using GB5009.9, the moisture content using GB5009.3, and the ash content using GB5009.4. In one embodiment of this invention, the collagen tripeptide has a protein content as high as 96.2%, a moisture content of 2.7%, and a ash content of 1.1%. Simultaneously, high-performance liquid chromatography (HPLC) is used to detect the content of protein components, and the collagen powder contains 3.8% GPH (Gly-Pro-Hyp) and 35.09% CTP (collagen threepeptide).

[0039] This invention also provides the application of the above-mentioned collagen tripeptide in the preparation of anti-aging products.

[0040] In one specific embodiment of the present invention, the in vitro antioxidant activity of the collagen tripeptide was evaluated, demonstrating that the collagen tripeptide possesses DPPH scavenging activity similar to that of vitamin C. In another embodiment of the present invention, a zebrafish model experiment was conducted on the collagen tripeptide. The MTC of the collagen tripeptide powder in the model zebrafish was 2000 and 250 μg / mL, respectively, indicating that the collagen tripeptide powder has significant ROS scavenging efficacy, β-galactosidase activity inhibition efficacy, and telomerase activity enhancement efficacy in the hydrogen peroxide-induced zebrafish aging model, exhibiting significant anti-aging effects.

[0041] The present invention also provides an anti-aging product, the active ingredient of which includes the above-mentioned collagen tripeptide, and also includes excipients.

[0042] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a collagen tripeptide, its preparation method, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1: Preparation of Collagen Tripeptide

[0044] Step 1. Add 12m to the enzymatic hydrolysis vessel 3 Deionized water was used, and the pH was adjusted to 8.8-9.0 with 1M sodium hydroxide. The temperature was raised to 80-85℃, and 1kg of heat-resistant protease (DuPont Danisco PHT) was added. The protease was thoroughly dissolved by stirring (60rpm). 1000kg of tilapia scales were prepared and subjected to high-temperature enzymatic hydrolysis pretreatment for 1h.

[0045] Step 2. Prepare 2 kg of alkaline protease (Xiasheng Industrial Group Co., Ltd., FDY-2241), add it to the enzymatic hydrolysis vessel in Step 1, stir thoroughly to dissolve, measure the pH and adjust the pH to 8.8-9.0 with 1M sodium hydroxide, stir thoroughly for 3 hours of enzymatic hydrolysis, then add 2 kg of neutral protease (Xiasheng Industrial Group Co., Ltd., FDG-223) and hydrolyze for 1 hour. Then raise the temperature to 100℃ for enzyme inactivation treatment.

[0046] Step 3. Filter the enzymatic hydrolysate from Step 2 using a ceramic membrane with a pore size of 0.2 μm at a pressure of 1.2 MPa and a flow rate of 2000 L / h. The ceramic membrane can effectively filter out large fat molecules, proteins, and other impurities, greatly improving the quality of the product. Then, filter the enzymatic hydrolysate through the ceramic membrane using an organic filter membrane with a pore size of 10 nm at a pressure of 2.3 MPa and a flow rate of 2800 L / h.

[0047] Step 4. Use a nanofiltration membrane with a molecular weight cutoff of 400 Da to cut off the flow and collect the permeate with a molecular weight of less than 400 Da. The permeate contains the main active peptides, mainly collagen tripeptides.

[0048] Step 5. Increase the concentration of the hydrolysate concentrated by high-pressure reverse osmosis membrane to 32%-36%, and then perform spray drying. The spray drying conditions are inlet air temperature of 135℃ and outlet air temperature of 75℃, finally obtaining collagen tripeptide.

[0049] Step 6. Physicochemical index detection of collagen tripeptide: protein content determination (GB5009.9), moisture content determination (GB5009.3), ash content determination (GB5009.4).

[0050] The protein content was found to be as high as 96.2%, and the moisture content was 2.7% and 1.1%, respectively.

[0051] Example 2: Detection of collagen tripeptides

[0052] The contents of GPH (gly-pro-hyp) and CTP (collagen threepeptide) in the collagen tripeptide prepared in Example 1 were detected.

[0053] 1. GPH testing method:

[0054] Testing equipment: Agilent 1260 high performance liquid chromatograph;

[0055] Chromatographic column: C18 hydrophilic column Zorbax SB-AQ, 5μm 4.6*250mm;

[0056] Mobile phase A: 0.1% trifluoroacetic acid + 99.9% ultrapure water; Mobile phase B: 0.1% trifluoroacetic acid + 99.9% acetonitrile;

[0057] Detection wavelength: 214 nm; detection time: 40 min; column temperature: 21℃~25℃; injection volume: 10 μL; flow rate: 0.8 ml / min.

[0058] Table 1. Liquid chromatography detection methods for collagen tripeptides

[0059]

[0060]

[0061] GPH standard liquid chromatography detection results Figure 1 As shown, the liquid chromatography detection results of collagen tripeptide are as follows: Figure 2 As shown, the GPH content in the collagen tripeptide was 3.8% as determined by testing.

[0062] 2. CTP test results:

[0063] Testing equipment: Waters 2489 high performance liquid chromatograph;

[0064] Chromatographic column: GE Sephadex G25 gel and column;

[0065] Mobile phase A: 0.01 mmol / L Tris (buffer solution pH 7.5) + 0.005 mol / L calcium chloride (anhydrous calcium chloride) + 0.15 mmol / L sodium chloride;

[0066] Detection wavelength: 214 nm; detection time: 90 min; column temperature: 21℃~25℃; injection volume: 10 μL; wavelength: 214 nm; flow rate: 0.3 ml / min.

[0067] CTP standard chromatographic detection results are as follows: Figure 3 As shown, the liquid chromatography detection results of collagen tripeptide CTP are as follows: Figure 4 As shown, the CTP content in collagen tripeptide was 35.09% as determined by testing.

[0068] Example 3: Evaluation of the in vitro antioxidant activity of collagen tripeptide

[0069] Determination of DPPH free radical scavenging ability

[0070] (1) The experiment was divided into a blank group, a negative control group, a test sample group and a positive control group;

[0071] (2) Preparation of DPPH free radical working solution: Prepare 1.00 g·L⁻¹ solution using anhydrous ethanol as solvent. -1DPPH radical stock solution was stored at 4°C protected from light for later use; 1 mL of DPPH radical stock solution was mixed with 9 mL of anhydrous ethanol to obtain a concentration of 100 mg·L⁻¹. - 1 DPPH free radical working solution.

[0072] Construction of the DPPH free radical standard curve: Dilute the DPPH free radical working solution sequentially with anhydrous ethanol to the desired concentration (mg·L⁻¹). -1 Sample solutions with concentrations of 100, 80, 60, 40, 20, 10, and 5 were prepared, with six replicates for each concentration. The absorbance at 517 nm was measured. A standard curve was plotted with DPPH radical concentration on the x-axis and absorbance on the y-axis, and the linear regression equation was obtained: y = 0.0079x + 0.039(R²). 2 =0.9999). This indicates that the concentration of DPPH free radicals is between 5 and 100 mg / L. -1 The linear relationship between absorbance and absorbance is good within the range.

[0073] Determination of DPPH free radical scavenging rate: Take an appropriate amount of collagen tripeptide and dilute it sequentially with anhydrous ethanol to a concentration (g·L). -1 Sample solutions with concentrations of 0.13, 0.25, 0.5, 1, 2, 4, and 8 were respectively mixed with 100 mg·L⁻¹. -1 The DPPH free radical working solution was mixed evenly at a volume ratio of 1:1 and reacted at 37°C in the dark for 30 min. The absorbance at 517 nm was measured. Three replicates were set up for each concentration, and VC of the same concentration was used as a positive control. The DPPH free radical scavenging rate was calculated according to formula (1):

[0074]

[0075] In the formula: A0 is the absorbance of the anhydrous ethanol + DPPH free radical working solution; A1 is the absorbance of the sample solution + DPPH free radical working solution; A2 is the absorbance of the sample solution + anhydrous ethanol.

[0076] The results are shown in Table 2, ranging from 0.13 to 8 mg / mL. -1 The ability of collagen tripeptide to scavenge DPPH free radicals within the dosage range was dose-dependent, especially when the collagen tripeptide concentration was 0.13 mg / mL. -1 The ability to scavenge DPPH free radicals was 22.38% at a collagen tripeptide concentration of 8 mg / mL. -1 The ability to scavenge DPPH free radicals was 89.2%, IC50. 50 The value was 0.61 mg·mL -1 When the mass concentration of vitamin C is 0.13 mg / mL -1 The ability to scavenge DPPH free radicals was 28.59% when vitamin C was at 8 mg / mL.-1 The ability to scavenge DPPH free radicals was 95.57%, and the IC 50 value was 0.25 mg·mL -1 ; From the results, it can be seen that the IC 50 value of collagen tripeptide for scavenging DPPH was relatively close to that of the vitamin C group, showing good DPPH scavenging activity.

[0077] Table 2 Scavenging ability of collagen tripeptide on DPPH free radicals

[0078]

[0079] Example 4 Zebrafish test on the anti-aging effect of collagen tripeptide

[0080] In this example, the statistical processing results were expressed as mean±SE. Statistical analysis was performed using SPSS 26.0 software, and p<0.05 indicated that the difference was statistically significant.

[0081] I. Evaluation of ROS scavenging efficacy

[0082] 1.1. Sample preparation information

[0083] The collagen tripeptide powder prepared in Example 1 was all prepared into a 20.0 mg / mL stock solution with standard dilution water and used immediately.

[0084] Positive control: Catalase, brown liquid, batch number K2010330, Shanghai Aladdin Biochemical Technology Co., Ltd., stored in the dark at -20°C. It was prepared into a 200 mg / mL stock solution with ultrapure water and stored in the dark at -20°C.

[0085] 1.2. Experimental animals

[0086] Zebrafish were all raised in fish-raising water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450 - 550 μS / cm; pH was 6.5 - 8.5; hardness was 50 - 100 mg / L CaCO3). The license number for the use of experimental animals was: SYXK(Zhe)2012 - 0171. The feeding management met the requirements of international AAALAC certification (certification number: 001458).

[0087] Wild-type AB strain zebrafish were used in the natural paired mating and breeding method. Zebrafish at 6 hours post-fertilization (6hpf) were used for the determination of the maximum test concentration (MTC) of the sample and the evaluation of ROS scavenging efficacy.

[0088] 1.3. Instruments, consumables and reagents

[0089] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (NestBiotech, China); 96-well microplate (Costar, China); Multifunctional microplate reader (SPARK, TECAN, Switzerland); CM-H2DCFDA (Catalog No. C6827, Life Technologies Corporation, USA).

[0090] Hydrogen peroxide (batch number G2023089, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0091] 1.4. MTC Measurement

[0092] Wild-type AB strain zebrafish (6 hpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Collagen tripeptide powder (concentration shown in Table 3) was administered in water, and a normal control group and a model control group were also included. Each well contained 3 mL of collagen tripeptide powder. Except for the normal control group, all other experimental groups were given hydrogen peroxide in water to establish a zebrafish aging model. During the sample treatment period, the number of dead zebrafish in each experimental group was counted daily and removed promptly. After 5 days of treatment at 28℃, the median toxicity (MTC) of the sample in the aging model zebrafish was determined. Under the experimental conditions, the MTC of collagen tripeptide powder in the model zebrafish was 2000 and 250 μg / mL, respectively.

[0093] Table 3. Results of the concentration exploration experiment for evaluating the anti-aging efficacy of samples (n=30)

[0094]

[0095]

[0096] 1.5. Evaluation of ROS removal efficacy

[0097] Wild-type AB strain zebrafish at 6 hpf were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Collagen tripeptide powder (concentration shown in Table 4) and catalase (positive control at 2000 μg / mL) were administered in water. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with hydrogen peroxide in water to establish a zebrafish aging model. After treatment with hydrogen peroxide for 4 dpf, reactive oxygen species (ROS) detection reagent CM-H2DCFDA was added to each experimental group. The zebrafish were then transferred to 96-well microplates, one zebrafish per well, with a volume of 100 μL per well, and incubated at 28℃ for 5 dpf. At the end of the experiment, the ROS fluorescence value of each experimental group was detected using a multi-functional microplate reader. The statistical analysis results of this index were used to evaluate the efficacy of the samples in scavenging ROS in the aging model zebrafish.

[0098] The ROS fluorescence value of the model control group (5041) was significantly lower than that of the normal control group (1107), with p < 0.001, indicating successful model establishment. The ROS fluorescence value of the positive control group (catalase at a concentration of 2000 μg / mL) was 2892, significantly lower than that of the model control group (p < 0.01), indicating a 55% ROS scavenging efficacy in zebrafish. This demonstrates that catalase has a significant ROS scavenging effect.

[0099] The ROS fluorescence values ​​of zebrafish in the collagen tripeptide powder concentration groups of 500, 1000, and 2000 μg / mL were 1034, 3895, and 3556, respectively, with ROS scavenging efficiencies of 102%, 29%, and 38%, respectively. Compared with the model control group, p<0.001, p>0.05, and p>0.05, respectively, indicating that collagen tripeptide powder has a significant ROS scavenging effect on the hydrogen peroxide-induced aging model of zebrafish under the experimental concentration conditions.

[0100] Table 4. Experimental results evaluating the ROS scavenging efficacy of the samples (n=10)

[0101]

[0102] In Table 4, compared with the model control group, **p<0.01, ***p<0.001.

[0103] II. Evaluation of the inhibitory efficacy against β-galactosidase activity

[0104] 2.1 Sample Preparation Information

[0105] The collagen tripeptide powder prepared in Example 1 was all prepared into a 20.0 mg / mL stock solution using standard dilution water and prepared fresh each time it was used.

[0106] Positive control: Catalase, brown liquid, batch number K2010330, Shanghai Aladdin Biochemical Technology Co., Ltd., stored in the dark at -20°C. Prepare a stock solution of 200 mg / mL with ultrapure water and store it in the dark at -20°C.

[0107] 2.2. Experimental animals

[0108] Zebrafish were all raised in fish-raising water at 28°C (water quality: add 200 mg of instant sea salt to every 1 L of reverse osmosis water, conductivity is 450 - 550 μS / cm; pH is 6.5 - 8.5; hardness is 50 - 100 mg / L CaCO3). The experimental animal use license number is: SYXK(Zhe)2012 - 0171. The feeding management meets the requirements of international AAALAC accreditation (accreditation number: 001458).

[0109] Wild-type AB strain zebrafish, reproduced by natural paired mating. Zebrafish at the age of 6 hpf were used to evaluate the inhibitory effect of the sample on β-galactosidase activity.

[0110] 2.3. Instruments, consumables and reagents

[0111] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (NestBiotech, China).

[0112] Hydrogen peroxide (batch number G2023089, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Cell senescence β-galactosidase staining kit (product number C0602, Beyotime Biotechnology, China); Methyl cellulose (batch number B2006074, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Tissue cell fixative; 4% tissue cell fixative (batch number 20201216, Beijing Solarbio Science & Technology Co., Ltd., China).

[0113] 2.4. Detection of β-galactosidase activity inhibition

[0114] Wild-type AB strain zebrafish (6 hpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Collagen tripeptide powder (concentration shown in Table 5) and catalase (positive control at 2000 μg / mL) were administered in water. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with hydrogen peroxide in water to establish a zebrafish aging model. After treatment with hydrogen peroxide for 5 dpf, the zebrafish were fixed overnight with 4% tissue cell fixative and stained using a β-galactosidase staining kit. After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Images were collected and analyzed using NIS-Elements D 3.20 advanced image processing software. The overall β-galactosidase staining intensity of the zebrafish was analyzed and statistically analyzed to evaluate the inhibitory effect of the samples on β-galactosidase activity in the aging model zebrafish.

[0115] The staining intensity of β-galactosidase in the model control group (51822 pixels) was significantly higher than that in the normal control group (47204 pixels), with p < 0.01, indicating successful model establishment. The staining intensity of β-galactosidase in the positive control group (2000 μg / mL catalase) was 46256 pixels, significantly lower than that in the model control group (p < 0.05), demonstrating an inhibitory effect of 121% on β-galactosidase activity in zebrafish. This indicates that catalase has an inhibitory effect on β-galactosidase activity.

[0116] The staining intensities of β-galactosidase in zebrafish at collagen tripeptide powder concentrations of 500, 1000, and 2000 μg / mL were 45809, 51491, and 53973 pixels, respectively, with inhibitory efficiencies of 130%, 7%, and -47% for β-galactosidase activity. Compared with the model control group, p<0.05, p>0.05, and p>0.05, respectively. The results are shown in Table 5. Figure 5 As shown, the collagen tripeptide powder has an inhibitory effect on β-galactosidase activity in a hydrogen peroxide-induced zebrafish aging model under the experimental concentration conditions.

[0117] Table 5. Results of zebrafish β-galactosidase staining intensity experiments after sample treatment (n=10)

[0118]

[0119] In Table 5, compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0120] III. Evaluation of the efficacy of telomerase activity enhancement

[0121] 3.1. Sample Preparation Information

[0122] The collagen tripeptide powder prepared in Example 1 was formulated into a 20.0 mg / mL stock solution with standard dilution water and used immediately after preparation.

[0123] Positive control: Catalase, brown liquid, batch number K2010330, Shanghai Aladdin Biochemical Technology Co., Ltd., stored in the dark at -20°C. It was formulated into a 200 mg / mL stock solution with ultrapure water and stored in the dark at -20°C.

[0124] 3.2. Experimental animals

[0125] Zebrafish were all raised in fish culture water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450 - 550 μS / cm; pH was 6.5 - 8.5; hardness was 50 - 100 mg / L CaCO3). The experimental animal use license number was: SYXK(Zhe)2012 - 0171. The feeding management met the requirements of international AAALAC certification (certification number: 001458).

[0126] Wild - type AB strain zebrafish were used for natural paired mating and reproduction. Zebrafish at 6 hpf were used for evaluating the efficacy of enhancing telomerase activity of the samples.

[0127] 3.3. Instruments, consumables and reagents

[0128] Dissecting microscope (SZX7, OLYMPUS, Japan), CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6 - well plate (NestBiotech, China); 96 - well plate (NestBiotech, China); Telomerase ELISA kit (batch number H21Y02, Shanghai Hengyuan Biotechnology Co., Ltd., China); High - speed centrifuge (TG16G, Shanghai Yihe Biotechnology Co., Ltd., China); Multifunctional microplate reader (SPARK, TECAN, Switzerland).

[0129] Hydrogen peroxide (batch number G2023089, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0130] 3.4. Telomerase activity detection method

[0131] Wild-type AB strain zebrafish (6 hpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Collagen tripeptide powder (concentration shown in Table 6) and catalase (positive control at 2000 μg / mL) were administered in water. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were treated with hydrogen peroxide in water to establish a zebrafish aging model. After treatment with hydrogen peroxide for 5 dpf, the zebrafish were homogenized, and the supernatant was collected. The reaction was performed using a telomerase ELISA kit, and the telomerase activity of each experimental group was measured using a multi-functional microplate reader. Statistical analysis of this index was used to evaluate the efficacy of the samples in enhancing telomerase activity in the aging model zebrafish.

[0132] The results are shown in Table 6. The telomerase activities of zebrafish in the collagen tripeptide powder concentration groups of 500, 1000 and 2000 μg / mL were 3.69, 3.64 and 2.95 IU / gprot, respectively, with telomerase activity enhancement effects of 45%, 43% and 16%, respectively. Compared with the model control group, p<0.01, p<0.01 and p>0.05, respectively, indicating that collagen tripeptide powder has the effect of promoting telomerase activity enhancement in the hydrogen peroxide-induced zebrafish aging model under the experimental concentration conditions.

[0133] Table 6. Telomerase activity in zebrafish after sample treatment (n=3)

[0134]

[0135] In Table 6, compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0136] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing collagen tripeptide, characterized in that, The process includes the following steps: (1) mixing an aqueous solution of thermostable protease with tilapia scales for high-temperature enzymatic hydrolysis pretreatment to obtain a pretreatment solution; the temperature of the high-temperature enzymatic hydrolysis pretreatment is 80~85℃; the thermostable protease is DuPont Danisco PHT; the pH value of the high-temperature enzymatic hydrolysis pretreatment is 8.8~9.0; and the time of the high-temperature enzymatic hydrolysis pretreatment is 1h~1.5h. (2) The pretreatment solution in step (1) is enzymatically hydrolyzed and then inactivated by alkaline protease and neutral protease respectively to obtain enzymatic hydrolysate; the enzymatic hydrolysis pH of alkaline protease is 8.8~9.0 and the enzymatic hydrolysis time is 3h~3.5h; the enzymatic hydrolysis time of neutral protease is 1h~1.5h. (3) The enzymatic hydrolysate is filtered sequentially using a ceramic membrane and an organic filter membrane to obtain the filtrate; (4) Use a nanofiltration membrane with a molecular weight cutoff of 400 Da to retain the permeate, which contains collagen tripeptide.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the thermostable protease and tilapia scales in step (1) is (0.8~1) kg: 1000 kg.

3. The preparation method according to claim 1, characterized in that, The ceramic membrane in step (3) has a pore size of 0.15~0.2μm and a processing pressure of 1.2~1.5MPa; The organic filter membrane has a pore size of 8~10nm and a processing pressure of 2.0~2.3MPa.

4. The preparation method according to claim 1, characterized in that, Step (4) involves collecting the permeate, followed by concentration and drying to obtain collagen tripeptide powder.

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