Collagen tripeptide as well as preparation method and application thereof

Abstract

The invention provides collagen tripeptide as well as a preparation method and application thereof, and belongs to the technical field of active peptide. The invention provides a preparation method of collagen tripeptide, which comprises the following steps: carrying out enzymolysis treatment on tilapia scales and high-temperature-resistant protease, then carrying out enzymolysis treatment by using alkaline protease and neutral protease in sequence, filtering enzymatic hydrolysate by using a ceramic membrane and an organic filter membrane in sequence, and intercepting by using a 400Da nanofiltration membrane to obtain collagen tripeptide. The permeate contains active peptide mainly composed of collagen tripeptide. After the permeate is concentrated and dried, the collagen tripeptide powder is detected, and the protein content reaches 96.2%, the GPH content is 3.8%, and the CTP content is 35.09%. The collagen tripeptide powder has better DPPH (1, 1-diphenyl-2-picrylhydrazyl) scavenging activity; the traditional Chinese medicine composition has an obvious ROS removing effect, a beta-galactosidase activity inhibiting effect and a telomerase activity enhancing promoting effect on a hydrogen peroxide induced zebrafish aging model, and shows an obvious anti-aging effect.

Description

Technical Field

[0001] The invention belongs to the technical field of active peptides, and specifically relates to a collagen tripeptide and a preparation method and application thereof. Background Art

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

[0003] At present, there are a large number of collagen-based anti-aging products on the market, among which products in the form of collagen peptides are the most common. Collagen peptides are peptides with smaller molecular weights obtained by enzymatic hydrolysis of collagen. They are easily absorbed by the human body and have multiple biological activities such as promoting collagen synthesis, anti-oxidation, and moisturizing. However, most traditional collagen peptide products are mixed peptides with a wide molecular weight distribution and unclear active ingredients, which affects the stability and reliability of their anti-aging effects.

[0004] In this context, collagen tripeptide, as an oligopeptide with a specific molecular weight (about 300-500Da), has gradually become the focus of anti-aging research due to its stable structure, easy absorption, and clear activity. Studies have shown that collagen tripeptide has significant antioxidant, anti-inflammatory and extracellular matrix synthesis promoting abilities, which can effectively slow down skin aging and improve joint health. However, the existing preparation methods of collagen tripeptide have problems such as low yield, low purity, and complex process, which limit its wide application in anti-aging products. Summary of the invention

[0005] The invention provides a collagen tripeptide and a preparation method and application thereof. The collagen tripeptide has high yield and 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 high temperature resistant protease with tilapia scales for high temperature enzymatic pretreatment to obtain a pretreated solution; the temperature of the high temperature enzymatic pretreatment is 80-85° C.;

[0007] (2) using alkaline protease and neutral protease to enzymatically hydrolyze the pretreatment solution of step (1) and then inactivate the enzymes to obtain an enzymatic hydrolyzate;

[0008] (3) filtering the enzymatic hydrolyzate using a ceramic membrane and an organic filter membrane in sequence to obtain a filtrate;

[0009] (4) A nanofiltration membrane with a molecular weight cutoff of 400 Da is used for interception, and the permeate is collected, wherein the permeate contains collagen tripeptide.

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

[0011] In a specific embodiment of the present invention, the mass ratio of the thermostable protease to the tilapia scale in step (1) is (0.8-1) kg:1000 kg.

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

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

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

[0015] The pore size of the organic filtration membrane is 8-10 nm, and the processing pressure is 2.0-2.3 MPa.

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

[0017] The present invention also provides collagen tripeptide prepared by the above preparation method.

[0018] The present invention also provides the use of the collagen tripeptide in the preparation of anti-aging products.

[0019] The present invention also provides an anti-aging product, the active ingredients of which include the above collagen tripeptide and auxiliary materials.

[0020] Beneficial effects: The present invention provides a method for preparing collagen tripeptide. Compared with the prior art, tilapia scales do not need to be pretreated, but the tilapia scales are directly enzymatically hydrolyzed with a high-temperature resistant protease, and then enzymatically hydrolyzed with an alkaline protease and a neutral protease in sequence, the enzymatic hydrolyzate is filtered with a ceramic membrane and an organic filter membrane in sequence, and then intercepted with a nanofiltration membrane, and a permeate with a molecular weight less than 400 Da is collected, and the permeate contains active peptides mainly composed of collagen tripeptide.

[0021] After the permeate is concentrated and dried, the collagen tripeptide powder is used for detection, and the protein content is as high as 96.2%, and the ash content is 1.1%; and the GPH (Gly-Pro-Hyp) content in the collagen tripeptide powder is 3.8%, and the CTP (collagen tripeptide) content is 35.09%.

[0022] The present invention evaluates the in vitro antioxidant activity of the prepared collagen tripeptide powder, and the IC value of the collagen tripeptide in scavenging DPPH is 50 The value is close to that of vitamin C, and it has good DPPH scavenging activity; at the same time, the collagen tripeptide powder was also tested on zebrafish, and the MTC of the collagen tripeptide powder for the model zebrafish was 2000 and 250 μg / mL, respectively, indicating that the collagen tripeptide powder has obvious ROS scavenging effect, β-galactosidase activity inhibition effect and telomerase activity enhancement effect on the zebrafish aging model induced by hydrogen peroxide, showing obvious anti-aging effect, so as to meet the current demand for high-quality active peptides. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 This is the liquid phase chromatogram of collagen tripeptide GPH;

[0025] Figure 3 This is the liquid chromatogram of CTP standard;

[0026] Figure 4 This is the liquid chromatogram of collagen tripeptide CTP detection;

[0027] Figure 5 This is a typical graph of zebrafish β-galactosidase staining intensity after sample treatment. The blue color in the figure represents the β-galactosidase staining intensity, and the darker the color, the stronger the activity. DETAILED DESCRIPTION

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

[0029] (2) using alkaline protease and neutral protease to enzymatically hydrolyze the pretreatment solution of step (1) and then inactivate the enzymes to obtain an enzymatic hydrolyzate;

[0030] (3) filtering the enzymatic hydrolyzate using a ceramic membrane and an organic filter membrane in sequence to obtain a filtrate;

[0031] (4) A nanofiltration membrane with a molecular weight cutoff of 400 Da is used for interception, and the permeate is collected, wherein the permeate contains collagen tripeptide.

[0032] When preparing the aqueous solution of the thermostable protease of the present invention, as in one embodiment, 12 m 3 Deionized water, using 1M sodium hydroxide to adjust the pH value to 8.8-9.0, heated to 80-85°C, and then adding 1kg of high temperature resistant protease (DuPont Danisco PHT), stirring and dissolving the protease to obtain the aqueous solution of the high temperature resistant protease. The rotation speed of the full stirring of the present invention can be 60rpm. The present invention mixes the aqueous solution of the high temperature resistant protease with tilapia scales, such as adding 1000kg of tilapia scales to the aqueous solution of the high temperature resistant protease in one embodiment, and pre-treating the solution with high temperature enzymolysis for 1h.

[0033] The present invention performs enzymatic hydrolysis on the pretreatment liquid, and the enzymatic hydrolysis includes sequentially performing 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, fully stirred and dissolved, and after measuring the pH, 1M sodium hydroxide is used to adjust the pH value to 8.8-9.0. After fully stirring and enzymolysis for 3 hours, 2 kg of neutral protease (Xiasheng Industrial Group Co., Ltd., FDG-223) is continued to be added, and after enzymolysis for 1 hour, the temperature is raised to 100°C for enzyme inactivation treatment.

[0034] The present invention filters the enzymatic solution after the enzyme inactivation, including using ceramic membrane filtration and organic filter membrane treatment in sequence. Wherein, when the ceramic membrane filtration treatment is performed, in a specific embodiment, the enzymatic solution is filtered with a ceramic membrane with a pore size of 0.2 μm, the treatment pressure is 1.2 MPa, and the flow rate is stabilized to 2000 L / h. The ceramic membrane can fully filter out fat macromolecular proteins and other impurities, greatly improving the quality of the product. The filtrate after the ceramic membrane filtration treatment is then filtered with an organic filter membrane, and the pore size of the organic filter membrane is 10 nm, the treatment pressure is 2.3 MPa, and the flow rate is stabilized at 2800 L / h. The ceramic membrane filtration of the present invention intercepts macromolecular impurities and proteins that are not hydrolyzed.

[0035] The present invention intercepts the filtrate after filtration by an organic filtration membrane. In a specific embodiment, a nanofiltration membrane with a cutoff molecular weight of 400Da (Dalton) is used for interception, and a permeate with a molecular weight less than 400Da is collected. The permeate contains a main active peptide mainly composed of collagen tripeptide.

[0036] The present invention concentrates the permeate, such as using a high-pressure reverse osmosis membrane to concentrate the liquid to increase the liquid concentration to 32% to 36%, and then spray-drying is performed. In one embodiment, the spray-drying conditions can be an inlet air temperature of 135°C and an outlet air temperature of 75°C, and finally collagen tripeptide powder is obtained.

[0037] The present invention also provides collagen tripeptide prepared by the above preparation method.

[0038] The present invention also performs physical and chemical index detection on the collagen tripeptide, including using the method of GB5009.9 to determine the protein content, using the method of GB5009.3 to determine the moisture content, and using the method of GB5009.4 to determine the ash content. In one embodiment of the present invention, the protein content of the collagen tripeptide is as high as 96.2%, the moisture content is 2.7%, and the moisture content is 1.1%; at the same time, the content of the components in the protein is detected by high performance liquid chromatography, and the GPH (Gly-Pro-Hyp) content in the collagen powder is 3.8%, and the CTP (collagen threepeptide) content is 35.09%.

[0039] The present invention also provides the use of the collagen tripeptide in the preparation of anti-aging products.

[0040] In a specific embodiment of the present invention, the collagen tripeptide was evaluated for its in vitro antioxidant activity, proving that the collagen tripeptide has DPPH scavenging activity similar to that of vitamin C. In another embodiment of the present invention, the collagen tripeptide was also subjected to a zebrafish model test, and the MTC of the collagen tripeptide powder on the model zebrafish was 2000 and 250 μg / mL, respectively, indicating that the collagen tripeptide powder has obvious ROS scavenging effect, β-galactosidase activity inhibition effect and telomerase activity enhancement effect on the zebrafish aging model induced by hydrogen peroxide, showing obvious anti-aging effect.

[0041] The present invention also provides an anti-aging product, the active ingredients of which include the above collagen tripeptide and auxiliary materials.

[0042] In order to further illustrate the present invention, a collagen tripeptide provided by the present invention and its preparation method and application are described in detail below in conjunction with examples, but they 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 3 Deionized water, adjust the pH to 8.8-9.0 with 1M sodium hydroxide, raise the temperature to 80-85°C, add 1kg of high temperature resistant protease (DuPont Danisco PHT), stir well to dissolve the protease (speed 60rpm) to prepare 1000kg of tilapia scales, and pre-treat with high temperature enzymatic hydrolysis for 1h;

[0045] Step 2. Prepare 2 kg of alkaline protease (Xiasheng Industrial Group Co., Ltd., FDY-2241), add it to the enzymolysis tank of step 1, stir it thoroughly to dissolve, measure the pH and adjust the pH to 8.8-9.0 with 1 M sodium hydroxide, stir it thoroughly for 3 hours, continue to add 2 kg of neutral protease (Xiasheng Industrial Group Co., Ltd., FDG-223), and then heat it to 100°C for enzyme inactivation after enzymolysis for 1 hour;

[0046] Step 3. The enzymatic hydrolysate of step 2 is filtered through a ceramic membrane with a pore size of 0.2 μm, the processing pressure is 1.2 MPa, and the flow rate is stabilized at 2000 L / h. The ceramic membrane can fully filter out fat macromolecular proteins and other impurities, greatly improving the quality of the product; the enzymatic hydrolysate filtered through the ceramic membrane is treated with an organic filter membrane with a pore size of 10 nm, the processing pressure is 2.3 MPa, and the flow rate is stabilized at 2800 L / h;

[0047] Step 4. Using a nanofiltration membrane with a molecular weight cutoff of 400 Da (Dalton) to intercept, collect the permeate with a molecular weight less than 400 Da, the permeate contains the main active peptides mainly composed of collagen tripeptide;

[0048] Step 5. The hydrolyzate is concentrated by a high-pressure reverse osmosis membrane to increase its concentration to 32% to 36%, and then spray-dried. The spray-drying conditions are an inlet air temperature of 135°C and an outlet air temperature of 75°C, and finally collagen tripeptide is obtained;

[0049] Step 6. Physical and chemical index detection of collagen tripeptide: determination of protein content (GB5009.9), determination of water content (GB5009.3), and determination of ash content (GB5009.4).

[0050] It was determined that the protein content was as high as 96.2%, the moisture content was 2.7%, and the water content was 1.1%.

[0051] Example 2 Detection of collagen tripeptide

[0052] The GPH (gly-pro-hyp) content and CTP (collagen three peptide) content in the collagen tripeptide prepared in Example 1 were detected.

[0053] 1. GPH detection 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: 214nm; detection time 40min; column temperature: 21℃~25℃, injection volume 10μL, flow rate 0.8ml / min.

[0058] Table 1 Collagen tripeptide liquid phase detection method

[0059]

[0060]

[0061] GPH standard liquid chromatography test results Figure 1 As shown, the results of collagen tripeptide liquid chromatography detection are as follows Figure 2 As shown, the content of GPH in collagen tripeptide was detected to be 3.8%.

[0062] 2. CTP test results:

[0063] Testing equipment: Waters2489 high performance liquid chromatograph;

[0064] Chromatographic column: GE sephadex G25 gel and chromatographic column;

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

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

[0067] The results of the chromatographic test of CTP standard are as follows Figure 3 As shown, the results of liquid chromatography detection of collagen tripeptide CTP are as follows Figure 4 As shown, the content of CTP in collagen tripeptide was detected to be 35.09%.

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

[0069] Determination of DPPH free radical scavenging ability

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

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

[0072] Drawing of DPPH free radical standard curve: DPPH free radical working solution was diluted with anhydrous ethanol to concentrations (mg·L -1 ) were 100, 80, 60, 40, 20, 10, and 5 sample solutions, and 6 replicates were set for each concentration. The absorbance at 517 nm was measured. A standard curve was drawn with the DPPH free radical concentration as the abscissa and the absorbance as the ordinate, and the linear regression equation was fitted: y = 0.0079x + 0.039 (R 2 =0.9999). This indicates that the DPPH free radical concentration is between 5 and 100 mg·L -1 The absorbance has a good linear relationship within this range.

[0073] Determination of DPPH free radical scavenging rate: Take an appropriate amount of collagen tripeptide and dilute it with anhydrous ethanol to a concentration (g·L -1 ) were 0.13, 0.25, 0.5, 1, 2, 4, and 8 respectively, and 100 mg·L -1 The DPPH free radical working solution was mixed evenly at a volume ratio of 1:1, reacted at 37°C in the dark for 30 minutes, and the absorbance at 517 nm was measured; 3 replicates were set 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] Wherein: A0 is the absorbance of anhydrous ethanol + DPPH free radical working solution; A1 is the absorbance of sample solution + DPPH free radical working solution; A2 is the absorbance of sample solution + anhydrous ethanol.

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

[0077] Table 2 Collagen tripeptide scavenging ability of DPPH free radicals

[0078]

[0079] Example 4 Anti-aging test of collagen tripeptide in zebrafish

[0080] In this embodiment, the statistical processing results are 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] 1. Evaluation of ROS scavenging efficacy

[0082] 1.1. Sample preparation information

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

[0084] Positive control: Catalase, brown liquid, batch number K2010330, Shanghai Aladdin Biochemical Technology Co., Ltd., stored at -20℃ away from light. Prepared into 200 mg / mL stock solution with ultrapure water, stored at -20℃ away from light.

[0085] 1.2. Experimental animals

[0086] Zebrafish were kept in 28°C fish water (water quality: 200 mg instant sea salt per 1L reverse osmosis water, conductivity 450-550 μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3), experimental animal use license number: SYXK (Zhejiang) 2012-0171. The feeding and management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0087] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 6 hours post fertilization (6 hpf) were used for the determination of maximum detection concentration (MTC) of samples 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 ELISA plate (Costar, China); multifunctional ELISA reader (SPARK, TECAN, Switzerland); CM-H2DCFDA (Cat. No. C6827, Life Technologies Corporation, USA).

[0090] Hydrogen peroxide (Batch No. G2023089, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0091] 1.4. MTC determination

[0092] 6hpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Collagen tripeptide powder (concentrations are shown in Table 3) was given in water, and a normal control group and a model control group were set up at the same time, with a capacity of 3mL per well. Except for the normal control group, hydrogen peroxide was given in water to establish a zebrafish aging model in all other experimental groups. During the sample treatment, the number of zebrafish deaths in each experimental group was counted every day and removed in time. After 5 days of treatment at 28°C, the MTC of the sample for the aging model zebrafish was determined. Under the experimental conditions, the MTC of collagen tripeptide powder for the model zebrafish was 2000 and 250μg / mL, respectively.

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

[0094]

[0095]

[0096] 1.5. Evaluation of ROS scavenging efficacy

[0097] 6hpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Collagen tripeptide powder (concentrations are shown in Table 4) was given in water, and the positive control catalase concentration was 2000μg / mL. A normal control group and a model control group were set up at the same time, with a capacity of 3mL per well. Except for the normal control group, hydrogen peroxide was given in water to establish a zebrafish aging model in all other experimental groups. After the samples were co-treated with hydrogen peroxide until 4dpf, the reactive oxygen species (ROS) detection reagent CM-H2DCFDA was added to each experimental group, and then the zebrafish were transferred to a 96-well ELISA plate, 1 tail per well, and the capacity of each well was 100μL. They were placed in a 28℃ incubator and continued to incubate until 5dpf. At the end of the experiment, the ROS fluorescence value of zebrafish in each experimental group was detected by a multifunctional ELISA instrument, and the statistical analysis results of this index were used to evaluate the ROS scavenging effect of the sample on the aging model zebrafish.

[0098] The ROS fluorescence value of zebrafish in the model control group (5041) was compared with that in the normal control group (1107), p<0.001, indicating that the model was successfully established. The ROS fluorescence value of zebrafish in the positive control catalase 2000μg / mL concentration group was 2892, p<0.01 compared with the model control group, and the ROS scavenging effect on zebrafish was 55%, indicating that catalase has a significant ROS scavenging effect.

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

[0100] Table 4 Sample ROS scavenging efficacy evaluation experimental results (n = 10)

[0101]

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

[0103] 2. Evaluation of β-galactosidase activity inhibition efficacy

[0104] 2.1. Sample preparation information

[0105] The collagen tripeptide powder prepared in Example 1 was prepared into a 20.0 mg / mL stock solution using standard dilution water and prepared immediately before use.

[0106] Positive control: Catalase, brown liquid, batch number K2010330, Shanghai Aladdin Biochemical Technology Co., Ltd., stored at -20℃ away from light. Prepared into 200 mg / mL stock solution with ultrapure water, stored at -20℃ away from light.

[0107] Experimental animals

[0108] Zebrafish were kept in 28°C fish water (water quality: 200 mg instant sea salt per 1L reverse osmosis water, conductivity 450-550 μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3), experimental animal use license number: SYXK (Zhejiang) 2012-0171. The feeding and management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0109] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish at the age of 6 hpf were used for the evaluation of the β-galactosidase activity inhibition efficacy of the samples.

[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 No. G2023089, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Cell senescence β-galactosidase staining kit (Cat. No. C0602, Beyotime Biotechnology, China); Methylcellulose (Batch No. B2006074, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Tissue cell fixative; 4% tissue cell fixative (Batch No. 20201216, Beijing Solebold Technology Co., Ltd., China).

[0113] 2.4. β-Galactosidase activity inhibition assay

[0114] 6hpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Collagen tripeptide powder (concentrations are shown in Table 5) was given in water, and the positive control catalase concentration was 2000μg / mL. A normal control group and a model control group were set up at the same time, with a capacity of 3mL per well. Except for the normal control group, all other experimental groups were given hydrogen peroxide in water to establish a zebrafish aging model. After the sample was co-treated with hydrogen peroxide to 5dpf, the zebrafish was fixed with 4% tissue cell fixative overnight and stained with a β-galactosidase staining kit. After staining, 10 zebrafish were randomly selected from each experimental group and placed under a dissecting microscope to take pictures and save the pictures. NIS-Elements D 3.20 advanced image processing software was used for image analysis and data collection. The overall β-galactosidase staining intensity of the zebrafish was analyzed and statistically analyzed. The statistical analysis results of this indicator were used to evaluate the inhibitory effect of the sample on the β-galactosidase activity of the aging model zebrafish.

[0115] The zebrafish β-galactosidase staining intensity in the model control group (51822 pixels) was compared with the normal control group (47204 pixels) p<0.01, indicating that the model was successfully established. The zebrafish β-galactosidase staining intensity in the positive control catalase 2000μg / mL concentration group was 46256 pixels, compared with the model control group p<0.05, and the inhibitory effect on zebrafish β-galactosidase activity was 121%, indicating that catalase has the inhibitory effect on β-galactosidase activity.

[0116] The zebrafish β-galactosidase staining intensity in the 500, 1000 and 2000 μg / mL concentration groups of collagen tripeptide powder was 45809, 51491 and 53973 pixels, respectively, and the β-galactosidase activity inhibition efficacy was 130%, 7% and -47%, respectively, compared with the model control group, p < 0.05, p > 0.05 and p > 0.05, the results are shown in Tables 5 and Figure 5 As shown, it is suggested that collagen tripeptide powder has the effect of inhibiting β-galactosidase activity in the zebrafish aging model induced by hydrogen peroxide under the experimental concentration conditions.

[0117] Table 5 Zebrafish β-galactosidase staining intensity experimental results 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] 3. Evaluation of the efficacy of enhancing telomerase activity

[0121] 3.1. Sample preparation information

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

[0123] Positive control: Catalase, brown liquid, batch number K2010330, Shanghai Aladdin Biochemical Technology Co., Ltd., stored at -20℃ away from light. Prepared into 200 mg / mL stock solution with ultrapure water, stored at -20℃ away from light.

[0124] 3.2 Experimental animals

[0125] Zebrafish were kept in 28°C fish water (water quality: 200 mg instant sea salt per 1L reverse osmosis water, conductivity 450-550 μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3), experimental animal use license number: SYXK (Zhejiang) 2012-0171. The feeding and management complies with the requirements of the international AAALAC certification (certification number: 001458).

[0126] Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish at the age of 6 hpf were used for the evaluation of the telomerase activity enhancement efficacy 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 (lot 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 No. G2023089, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0130] Telomerase activity detection method

[0131] 6hpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Collagen tripeptide powder (concentrations are shown in Table 6) was given in water, and the positive control catalase concentration was 2000μg / mL. A normal control group and a model control group were set up at the same time, and the capacity of each well was 3mL. Except for the normal control group, hydrogen peroxide was given in water to establish a zebrafish aging model in all other experimental groups. After the sample was co-treated with hydrogen peroxide until 5dpf, the supernatant of the zebrafish homogenate was taken, and the telomerase ELISA kit was used for reaction. The telomerase activity of each experimental group was determined by a multifunctional microplate reader, and the statistical analysis results of this index were used to evaluate the sample's efficacy in enhancing the telomerase activity of the aging model zebrafish.

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

[0133] Table 6 Zebrafish telomerase activity 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 embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, 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 method comprises the following steps: (1) mixing an aqueous solution of a high temperature resistant protease with tilapia scales for high temperature enzymatic pretreatment to obtain a pretreated solution; the temperature of the high temperature enzymatic pretreatment is 80-85° C.; (2) using alkaline protease and neutral protease to enzymatically hydrolyze the pretreatment solution of step (1) and then inactivate the enzymes to obtain an enzymatic hydrolyzate; (3) filtering the enzymatic hydrolyzate using a ceramic membrane and an organic filter membrane in sequence to obtain a filtrate; (4) A nanofiltration membrane with a molecular weight cutoff of 400 Da is used for interception, and the permeate is collected, wherein the permeate contains collagen tripeptide.

2. The preparation method according to claim 1, characterized in that: The pH value of the high temperature enzymatic pretreatment in step (1) is 8.8 to 9.0, and the time of the high temperature enzymatic pretreatment is 1 h to 1.5 h.

3. The preparation method according to claim 1 or 2, characterized in that: The mass ratio of the high temperature resistant protease to the tilapia scale in step (1) is (0.8-1) kg:1000 kg.

4. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis pH value of the alkaline protease in step (2) is 8.8-9.0, and the enzymatic hydrolysis time is 3h-3.5h.

5. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis time of the neutral protease in step (2) is 1 h to 1.5 h.

6. The preparation method according to claim 1, characterized in that: The pore size of the ceramic membrane in step (3) is 0.15-0.2 μm, and the treatment pressure is 1.2-1.5 MPa; The pore size of the organic filtration membrane is 8-10 nm, and the processing pressure is 2.0-2.3 MPa.

7. The preparation method according to claim 1, characterized in that: After collecting the permeate in step (4), the process further comprises concentrating and drying to obtain collagen tripeptide powder.

8. Collagen tripeptide prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the collagen tripeptide according to claim 8 in the preparation of anti-aging products.

10. An anti-aging product, characterized in that: The active ingredient comprises the collagen tripeptide as claimed in claim 8, and also comprises auxiliary materials.

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