Collagen tripeptide powder as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of active ingredient extraction, and particularly relates to collagen tripeptide powder as well as a preparation method and application thereof. The preparation method of the collagen tripeptide comprises the following steps: (1) enzymolysis extraction: mixing fish scales and water, heating and stirring, adjusting the pH value, and adding a compound enzyme for enzymolysis; adding flavor enzyme for enzymolysis, boiling and inactivating to obtain protein enzymatic hydrolysate; (2) separation and purification: filtering and separating the protein enzymatic hydrolysate obtained in the step (1) to obtain filtrate; and (3) concentrating and drying: concentrating and drying the filtrate obtained in the step (2) to obtain collagen tripeptide powder. According to the collagen tripeptide powder prepared by the preparation method provided by the invention, no organic solvent or toxic and harmful compound is used in the preparation method, and the collagen tripeptide powder is simple and convenient to operate, safe, high in yield, low in cost, stable in product quality and environment-friendly.

Description

Technical Field

[0001] This invention belongs to the field of active ingredient extraction technology, specifically relating to a collagen tripeptide powder, its preparation method, and its application. Background Technology

[0002] Skin damage repair is a complex biological process, encompassing the inflammatory, proliferative, and remodeling phases. Collagen plays a particularly crucial role in the remodeling phase, during which fibroblasts synthesize new collagen, forming a new extracellular matrix and promoting wound healing. However, large collagen molecules are difficult for the skin to absorb, resulting in low bioavailability. Collagen is a triple helix structure composed of three α-peptide chains, exhibiting a highly stable molecular structure with a relative molecular weight exceeding 30 kDa, hindering direct absorption by the human body. Collagen tripeptides, short peptides composed of three amino acids linked by peptide bonds, typically have a molecular weight between 200-500 Daltons. They can directly penetrate the basal layer of the skin and have good affinity with surrounding tissues, assisting cells in producing collagen and promoting normal skin cell growth. Traditional collagen tripeptide extraction methods suffer from low efficiency, high cost, and low purity. Existing technologies require multiple filtration steps, and depending on the specific circumstances, each filtration step needs to be repeated to ensure solution purity. This makes the entire production process cumbersome and time-consuming. Furthermore, some collagen tripeptides lack research on their skin damage repair effects after preparation, hindering further optimization of the collagen tripeptide extraction process.

[0003] For example, Chinese patent CN109136317A discloses a method for extracting collagen peptides, including the following steps: (1) taking clean fish scales and performing enzymatic hydrolysis with an enzymatic hydrolysate at a temperature of 12°C; (2) filtering the hydrolysate after enzymatic hydrolysis to obtain collagen peptide liquid, which is then dried to obtain collagen peptide powder; the filtration accuracy range is 5μm; the enzymatic hydrolysis time is 20 hours, the pH value of the hydrolysate is 3-9, and the enzyme content in the hydrolysate is 1-5%; the enzyme is a mixture of bromelain, pepsin, and papain, and the mass ratio of bromelain, pepsin, and papain is 1:1:1; the amount of hydrolysate is 8 times the weight of the fish scales; the fish scales are fresh or dried. This invention extracts collagen peptides under low-temperature conditions, which maximizes the preservation of their bioactivity. Using specific enzymes, the hydrolyzed product is easily absorbed by the human body.

[0004] For example, Chinese patent CN114957386A discloses a method for producing collagen tripeptides from fish scales, belonging to the field of fish collagen peptide preparation technology. This invention uses a pretreatment solution composed of acetic acid, citric acid, EDTA, and microcrystalline cellulose for soaking. The efficient removal of impurities by the pretreatment solution provides a foundation for subsequent efficient enzymatic hydrolysis. Then, a two-stage enzymatic hydrolysis is performed using alkaline protease, neutral protease, and collagenase to improve hydrolysis efficiency. Simultaneously, to stimulate protease activity, this invention adds sodium citrate and dodecyl ethoxysulfonate to prepare active nano-calcium. The three components work synergistically to effectively stimulate protease activity, achieving complete enzymatic hydrolysis of collagen and significantly reducing the molecular weight of the enzyme product. Finally, membrane concentration and extraction are performed to further improve purity and reduce the product molecular weight. After freeze-drying, a high-purity collagen peptide product is obtained. The preparation process is simple, efficient, easy to operate, and produces a fine product.

[0005] Therefore, there is an urgent need to study a simple, high-yield, low-cost, stable product quality, and environmentally friendly method for the extraction and preparation of collagen tripeptides. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to provide a collagen tripeptide powder, its preparation method, and its applications. The method provided by this invention involves enzymatic extraction of degreased and delimed tilapia scales, separation and purification, concentration, and drying to obtain collagen tripeptide powder. The preparation process does not use any organic solvents or toxic or harmful compounds, and is simple, safe, yields high output, has low cost, provides stable product quality, and is environmentally friendly.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, the present invention provides a method for preparing collagen tripeptides, comprising the following steps: (1) Enzymatic extraction: Mix fish scales and water, heat and stir, adjust pH, add compound enzyme for enzymatic hydrolysis; then add flavor enzyme for enzymatic hydrolysis, boil to inactivate, and obtain proteolytic hydrolysate; (2) Separation and purification: The protein hydrolysate obtained in step (1) is filtered and separated to obtain the filtrate; (3) Concentration and drying: The filtrate obtained in step (2) is concentrated and dried to obtain collagen tripeptide powder.

[0008] Preferably, the weight ratio of fish scales to water in step (1) is 1:10-20, more preferably 1:20.

[0009] Preferably, the fish scales mentioned in step (1) are tilapia scales that have been degreased and de-ashed.

[0010] Preferably, the heating temperature in step (1) is 40-60°C, more preferably 40°C.

[0011] Preferably, the pH adjustment in step (1) refers to adjusting the pH to 6.5-7.5 using a 10% v / v dilute hydrochloric acid solution, more preferably 7.2.

[0012] Preferably, the amount of the compound enzyme added in step (1) is 0.5-0.6% of the weight of the fish scales.

[0013] Preferably, the complex enzyme in step (1) is composed of bromelain, alkaline protease and papain, with a mass ratio of 0.5:0.5-1:0.5-1, more preferably 0.5:1:1.

[0014] Preferably, the bromelain has an enzyme activity ≥2×10⁻⁶. 5 u / g; alkaline protease activity ≥2×10 5 u / g; Papain enzyme activity ≥2×10 5 u / g.

[0015] More preferably, the ratio of the enzyme activities of bromelain, alkaline protease and papain is 0.5:0.5-1:0.5-1, and more preferably 1:1:1.

[0016] Preferably, the flavor enzyme in step (1) is composed of bromelain and papain, with a mass ratio of 1:0.5-1, more preferably 1:1.

[0017] Preferably, the bromelain has an enzyme activity ≥2×10⁻⁶. 5 u / g; Papain enzyme activity ≥2×10 5 u / g; the ratio of enzyme activity of bromelain and papain is 1:0.5-1.

[0018] Preferably, the mass ratio of the compound enzyme to the flavor enzyme is 3-6:1, more preferably 5:1.

[0019] Preferably, the boiling inactivation temperature in step (1) is 95-100℃, more preferably 100℃.

[0020] Preferably, the filtration in step (2) involves first filtering with a microfiltration membrane with a pore size of 0.1-0.5 µm to obtain a filtrate, and then treating the filtrate with a 5000-10000 Dalton ultrafiltration membrane.

[0021] More preferably, the filtration in step (2) is to first use a microfiltration membrane with a pore size of 0.5 µm to obtain a filtrate, and then treat the filtrate with a 5000 Dalton ultrafiltration membrane.

[0022] Preferably, the concentration temperature in step (3) is 40-60°C, and the concentration is carried out until the mass fraction of solid content is 10-20%.

[0023] More preferably, the concentration temperature in step (3) is 55°C, and the concentration is carried out to a solid content of 15% by mass.

[0024] Preferably, the drying in step (3) is spray drying, and the inlet temperature of the spray drying is 160-200℃, more preferably 160℃.

[0025] On the other hand, the present invention also provides collagen tripeptide powder prepared by the above preparation method, wherein the collagen tripeptide powder contains more than 99% by mass of peptides with a relative molecular weight of less than 10,000 Daltons and more than 45% by mass of peptides with a relative molecular weight of 189-500 Daltons, and the detection method for the relative molecular weight distribution of the peptides is performed in accordance with the determination method in Appendix A of GB 31645-2018.

[0026] Preferably, the collagen tripeptide powder prepared by the above method contains 45-50% by mass of peptides with a mass of 189-500 Daltons.

[0027] Finally, the present invention also provides the application of the collagen tripeptide powder prepared by the above preparation method in the preparation of food, health food, medicine or cosmetics.

[0028] Specifically, the application is used to treat or prevent a range of symptoms caused by an excess of free radicals.

[0029] Specifically, the dosage form of the medicine is any one of tablets, capsules, powders, granules, or oral liquids.

[0030] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0031] Specifically, the food and health products mentioned also include nutritionally acceptable excipients.

[0032] Specifically, the pharmaceutically acceptable excipients are excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers.

[0033] Compared with the prior art, the present invention has the following beneficial effects: (1) The preparation method provided by the present invention obtains collagen tripeptide powder by enzymatic extraction of tilapia scales that have been degreased and deashed, separation and purification, concentration and drying. No organic solvents or toxic and harmful compounds are used in the preparation process. The method is simple to operate, safe, has a high yield, low cost, stable product quality and is environmentally friendly, making it suitable for large-scale production.

[0034] (2) The collagen tripeptide powder prepared by the preparation method of the present invention has a good effect on promoting the repair of skin keratinocyte damage and ultraviolet damage. Attached Figure Description

[0035] Figure 1 Scratch diagrams showing how collagen tripeptide powder prepared in each embodiment and comparative example promotes the formation of keratinocytes in the skin; Figure 2 The collagen tripeptide powder prepared in each example and comparative example promotes the scratch repair rate of skin keratinocytes (compared with the blank control group, ****p<0.0001, ***p<0.001, ns represents no significant difference). Figure 3 The diagram shows the effect of collagen tripeptide powder prepared in each embodiment and comparative example on promoting the repair of ultraviolet-induced zebrafish tail fin damage. Figure 4 The collagen tripeptide powder prepared in each example and comparative example promotes the UV-induced dorsal fin area of ​​zebrafish (compared with the model, ****p<0.0001, **p<0.01, *p<0.05, ns represents no significant difference). Detailed Implementation

[0036] To make the inventive objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described in this specification are merely for explaining the invention and are not intended to limit the invention.

[0037] For simplicity, this invention only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0038] In the description of this invention, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "multiple" in "one or more" means two or more.

[0039] The above description of the invention is not intended to describe every disclosed embodiment or implementation of the invention. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the invention, guidance is provided through a series of embodiments, which can be used in various combinations. In each example, the examples are listed only as representative groups and should not be construed as exhaustive.

[0040] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the materials and reagents used are commercially available.

[0041] The sources of raw materials used in the following examples and comparative examples are shown in Table 1 below: Table 1

[0042] Example 1: A method for preparing collagen tripeptide, specifically comprising the following steps: (1) Enzymatic extraction: 100 kg of defatted and deashed dried tilapia scales were placed in a mixing tank. Bromelain, alkaline protease and papain were mixed in a mass ratio of 0.5:1:1 to form a compound enzyme. Bromelain and papain were mixed in a mass ratio of 1:1 to form a flavor enzyme. The mixture was set aside. Fish scales and water were mixed in a weight ratio of 1:20 and heated to 40°C. The pH was adjusted to 7.2 with 10% v / v dilute hydrochloric acid solution. 500 g of the mixed compound enzyme was added and the mixture was stirred for 2 h. 100 g of flavor enzyme was added and the mixture was stirred for 2 h. The enzyme was boiled at 95°C-100°C for 10 min to inactivate the enzyme and obtain the protein hydrolysate.

[0043] (2) Separation and purification: The protein hydrolysate was filtered through a ceramic membrane with a pore size of 0.5 µm, and the filtrate was then treated with a 5000 Dalton ultrafiltration membrane to obtain the filtrate; (3) Concentration and drying: Concentrate at 55°C to a solid content of 15wt%, then spray dry (inlet temperature is 160°C) to obtain 85.3kg collagen tripeptide powder (T-1), with a yield of 85.3wt%.

[0044] Using the detection method in Appendix A of GB 31645-2018, the proportion of collagen peptides with a relative molecular weight of less than 10,000 Daltons was found to be 99.9%, and collagen tripeptides (189-500 Daltons) accounted for 48.6% of all peptides.

[0045] Example 2: A method for preparing collagen tripeptide, specifically comprising the following steps: (1) Enzymatic extraction: 100 kg of defatted and deashed dried tilapia scales were placed in a mixing tank. Bromelain, alkaline protease and papain were mixed in a mass ratio of 0.5:0.5:0.5 to form a compound enzyme. Bromelain and papain were mixed in a mass ratio of 1:0.5 to form a flavor enzyme. The mixture was set aside. Fish scales and water were mixed in a weight ratio of 1:15 and heated to 60°C. The pH was adjusted to 6.5 with 10% v / v dilute hydrochloric acid solution. 500 g of the mixed compound enzyme was added and the mixture was stirred for 2 h. 150 g of flavor enzyme was added and the mixture was stirred for 2 h. The mixture was boiled at 95°C-100°C for 10 min to inactivate the enzyme and obtain the protein hydrolysate.

[0046] (2) Separation and purification: The protein hydrolysate was filtered through a ceramic membrane with a pore size of 0.1 µm, and the filtrate was then treated with a 10000 Dalton ultrafiltration membrane to obtain the filtrate; (3) Concentration and drying: Concentrate at 55°C to a solid content of 10wt%, then spray dry (inlet temperature of 200°C) to obtain 86.7kg collagen tripeptide powder (T-2), with a yield of 86.7wt%.

[0047] Using the detection method in Appendix A of GB 31645-2018, the proportion of collagen peptides with a relative molecular weight of less than 10,000 Daltons was found to be 99.9%, and collagen tripeptides (189-500 Daltons) accounted for 47.9% of all peptides.

[0048] Example 3: A method for preparing collagen tripeptide, specifically comprising the following steps: (1) Enzymatic extraction: 100 kg of defatted and deashed dried tilapia scales were placed in a mixing tank. Bromelain, alkaline protease and papain were mixed in a mass ratio of 0.5:0.5:0.5 to form a compound enzyme. Bromelain and papain were mixed in a mass ratio of 1:0.5 to form a flavor enzyme. The mixture was set aside. Fish scales and water were mixed in a weight ratio of 1:10 and heated to 40°C. The pH was adjusted to 6.5 with 10% v / v dilute hydrochloric acid solution. 600 g of the mixed compound enzyme was added and the mixture was stirred for 2 h. 100 g of flavor enzyme was added and the mixture was stirred for 2 h. The mixture was boiled at 95°C-100°C for 10 min to inactivate the enzyme and obtain the protein hydrolysate.

[0049] (2) Separation and purification: The protein hydrolysate was filtered through a ceramic membrane with a pore size of 0.2µm, and the filtrate was then treated with a 5000 Dalton ultrafiltration membrane to obtain the filtrate; (3) Concentration and drying: Concentrate at 60°C to a solid content of 20wt%, then spray dry (inlet temperature is 180°C) to obtain 84.8kg collagen tripeptide powder (T-3), with a yield of 84.8wt%.

[0050] Using the detection method in Appendix A of GB 31645-2018, the proportion of collagen peptides with a relative molecular weight of less than 10,000 Daltons was found to be 99.9%, and collagen tripeptides (189-500 Daltons) accounted for 48.9% of all peptides.

[0051] Comparative Example 1 The difference from Example 1 is that fish scales and water were mixed in a weight ratio of 1:8, while the rest was the same as in Example 1. 68.6 kg of collagen tripeptide powder (DT-1) was obtained, with a yield of 68.6 wt%.

[0052] Using the detection method in Appendix A of GB 31645-2018, the proportion of collagen peptides with a relative molecular weight of less than 10,000 Daltons was found to be 99.1%, and collagen tripeptides (189-500 Daltons) accounted for 13.5% of all peptides.

[0053] Comparative Example 2 The difference from Example 1 is that the heating temperature in step (1) was adjusted to 70°C, while the rest was the same as in Example 1. 63.6 kg of collagen tripeptide powder (DT-2) was obtained, with a yield of 63.6 wt%.

[0054] Using the detection method in Appendix A of GB 31645-2018, the proportion of collagen peptides with a relative molecular weight of less than 10,000 Daltons was found to be 99.1%, and collagen tripeptides (189-500 Daltons) accounted for 18.5% of all peptides.

[0055] Comparative Example 3 The difference from Example 1 is that alkaline protease was replaced with pepsin; otherwise, it was the same as Example 1. 80.1 kg of collagen tripeptide powder (DT-3) was obtained, with a yield of 80.1 wt%.

[0056] Using the detection method in Appendix A of GB 31645-2018, the proportion of collagen peptides with a relative molecular weight of less than 10,000 Daltons was found to be 78.5%, and collagen tripeptides (189-500 Daltons) accounted for 7.6% of all peptides.

[0057] Comparative Example 4 The difference from Example 1 is that the mass ratio of bromelain, alkaline protease, and papain in the complex enzyme is 2:1:1, while the rest is the same as in Example 1. 81.2 kg of collagen tripeptide powder (DT-4) was obtained, with a yield of 81.2 wt%.

[0058] Using the detection method in Appendix A of GB 31645-2018, the proportion of collagen peptides with a relative molecular weight of less than 10,000 Daltons was found to be 83.6%, and collagen tripeptides (189-500 Daltons) accounted for 25.4% of all peptides.

[0059] Effect Experiment 1. Cell scratch damage repair experiment The cell line used in this experiment was human immortalized keratinocytes (hereinafter referred to as Hacat cells), purchased from Wuhan Pronosei Technology Co., Ltd. The PBS, MEM basal medium, and fetal bovine serum used for cell culture were also purchased from Wuhan Pronosei Technology Co., Ltd. The instruments and equipment used included an electronic balance (0.1 g / L, METTLER, Switzerland), a bright-field microscope (MZ62, Mshot, China), a clean bench (Zhongke Meiling, China), and a carbon dioxide incubator (Bolu (Zhichu) PU-90A, China). The specific operating procedures are as follows. (1) Seed Hacat cell suspension in 12-well plates and incubate at 37°C in a 5% CO2 incubator until the cell density reaches 95%-100%; (2) After the cell density reaches 95%-100%, scratching and drug administration will begin. Since the collagen tripeptide powder prepared by this invention has good water solubility, it can be completely dissolved in cell culture medium without the need for solubilizing. Before scratching, prepare 10 mg / mL of collagen tripeptide powder test sample stock solution in each example and comparative example using serum-free MEM culture medium. Ultraviolet irradiation is required to remove microorganisms in the raw materials to avoid contamination. After filtration, the test sample stock solution is diluted to 1 mg / mL with fresh culture medium before drug administration. (3) Discard the old culture medium and use the tip of a 200μL pipette to make scratches perpendicular to the cell well plate, making three vertical scratches in each well; (4) Wash with PBS 1-2 times to remove cell debris clumps caused by the scratches; (5) Place the cell well plate under a microscope to observe and photograph the initial scratches; (6) Set up a blank control group (MEM medium without serum), a positive control group (MEM medium containing 10% serum), and a 1 mg / mL sample group to be tested. Add 1 mL to a 12-well plate (2 mL to a 6-well plate). After adding the drug, place the plate in a 37°C, 5% CO2 incubator for 24 hours. (7) Remove the culture medium from each well, observe and photograph the scratches under a microscope 24 hours after drug administration, such as... Figure 1 As shown; (8) ImageJ software analyzes the width of each scratch and calculates the scratch repair rate. Scratch repair rate = (S0H -S 24H ) / S 0H ×100%, where: S 0H S represents the scratch length after 0 hours. 24H The scratch length represents 24 hours. Results are expressed as mean ± standard deviation (mean ± SD), plotted using Graphpad Prism 8.0, and statistical analysis was performed. The results are shown in Table 2 and... Figure 2 As shown.

[0060] Table 2

[0061] Scratch repair experiment results are as follows Figure 1 and Figure 2 As shown in Table 2, the quantitative statistical results of scratch repair rate are presented. Table 2 shows that, compared to the blank control group, the collagen tripeptide powder prepared by the method of this invention in Examples 1, 2, and 3 promoted the repair of scratches on human skin keratinocytes, with p values ​​all < 0.001, indicating significant efficacy. However, the collagen tripeptide powders in Comparative Examples 1, 2, 3, and 4 did not significantly promote scratch repair, with p values ​​all > 0.05. This may be due to the lower peptide content and larger molecular weight.

[0062] 2. Zebrafish UV Damage Repair Experiment The wild-type AB strain zebrafish broodstock used in this experiment were purchased from Huante Biotechnology Co., Ltd., and the juveniles were bred by our company using the broodstock. The instruments and equipment used included a biochemical incubator (HT-250H-T, Huante Biotechnology, China), an electronic balance (0.1 g / L, METTLER, Switzerland), a six-well culture plate (corning, USA), a stereomicroscope (MZ62, Mshot, China), a microscope imaging system (BX53, OLYMPUS Japan), and an ultraviolet crosslinker (SCIENTZ03-II, SpectraMax Mini). 2.1 Determination of maximum tolerated concentration (1) Wild-type AB strain zebrafish embryos with normal development, 3-5 days after fertilization, were randomly selected and placed in a six-well culture plate, 30 embryos per well. The standard dilution water of the six-well plate was removed without harming the embryos. 3 mL of collagen tripeptide powder dilution prepared in each example and comparative example at concentrations of 20 mg / mL, 10 mg / mL, 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL was added to each well. The culture plate was covered and wrapped with aluminum foil. It was incubated in a biochemical incubator at (28.5±1)℃ in the dark for 24 hours. After 24 hours, the number of zebrafish that died in each concentration group was counted, and the maximum tolerated concentration of the sample for zebrafish was determined. The concentration at which no zebrafish died was taken as the maximum tolerated concentration of the sample for zebrafish.

[0063] (2) Modeling of ultraviolet damage in zebrafish and sample processing The experimental groups were set as a blank control group, a UV model group, and sample treatment groups (Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4). The blank control group did not undergo UV damage treatment (the individual wells were not placed in the UV crosslinker for irradiation, or the blank control wells were covered with aluminum foil to avoid UV exposure). The UV model group and sample treatment groups were placed in the UV crosslinker for fixed-intensity UV irradiation. The blank control group and UV model group were treated with purified water. Maximum tolerance concentration testing showed that the collagen tripeptide powder prepared in this invention had low toxicity to zebrafish; the survival rate of zebrafish treated with 10 mg / mL collagen tripeptide powder reached 100%. Therefore, the effective concentration of collagen tripeptide powder in subsequent experiments was set at 10 mg / mL. The sample treatment groups were treated with the samples to be tested before UV irradiation for 24 hours. (3) The conditions for modeling ultraviolet damage were: intensity of 0.1 J / cm. 2 UVB, intermittent irradiation, with a 30-minute interval between each irradiation, for a total of 6 irradiations; (4) After irradiation 6 times, zebrafish in each experimental group were observed and photographed under a bright-field microscope. At least 10 zebrafish were randomly selected from each experimental group, fixed with 3% methylcellulose, and the dorsal fin area of ​​the zebrafish was photographed and collected. When taking pictures, the zebrafish were positioned with their tails facing right and their backs facing upwards. The dorsal fin of the zebrafish tail was photographed with a 10x objective lens (100x magnification). The light source was adjusted to make the dorsal fin clear and easy to analyze later. The results are as follows. Figure 3 As shown.

[0064] (5) The dorsal fin area (A) of zebrafish was quantitatively analyzed using ImageJ software. The statistical analysis results of this index were used to evaluate the repair capacity of the samples against UVB-induced ultraviolet damage. Statistical results are expressed as mean ± SD. Graphpad Prism 8.0 software was used for plotting and statistical analysis. p < 0.05 indicated that the difference was statistically significant.

[0065] (6) Result determination: Using the dorsal fin area of ​​zebrafish as an indicator, a one-way ANOVA was performed with the results of the UV model group. If p < 0.05, the sample was determined to have the effect of promoting UV damage repair. The test results are shown in Table 3 and Figure 4 As shown.

[0066] Table 3

[0067] like Figure 3 As shown, compared with the blank control group, the dorsal fin area of ​​zebrafish in the UV model group showed significant shrinkage, indicating that the UV damage model was successfully established. Figure 4 As shown in Table 3, compared with the UV model group, the dorsal fin area of ​​zebrafish pretreated with collagen tripeptide powder in Examples 1, 2, and 3 was significantly larger, indicating that the collagen tripeptide powder prepared in this invention has a significant repair effect on UV-induced damage. Although Comparative Examples 1, 2, and 4 also have a certain effect on promoting the repair of UV damage in zebrafish, the effect is far less than that of Examples 1, 2, and 3.

[0068] In summary, the collagen tripeptide powder of the present invention has the effect of promoting the repair of scratches on human skin keratinocytes and promoting the repair of ultraviolet damage.

[0069] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing collagen tripeptide, characterized in that, Includes the following steps: (1) Enzymatic extraction: Mix fish scales and water, heat and stir, adjust pH, and add compound enzyme for enzymatic hydrolysis; Add flavor enzymes for enzymatic hydrolysis, boil to inactivate, and obtain proteolytic hydrolysate; (2) Separation and purification: The protein hydrolysate obtained in step (1) is filtered and separated to obtain the filtrate; (3) Concentration and drying: The filtrate obtained in step (2) is concentrated and dried to obtain collagen tripeptide powder; In step (1), the weight ratio of fish scales to water is 1:10-20.

2. The preparation method according to claim 1, characterized in that, The amount of the compound enzyme added in step (1) is 0.5-0.6% of the weight of the fish scales.

3. The preparation method according to claim 1, characterized in that, The heating temperature in step (1) is 40-60℃.

4. The preparation method according to claim 1, characterized in that, The complex enzyme described in step (1) consists of bromelain, alkaline protease and papain, with a mass ratio of 0.5:0.5-1:0.5-1.

5. The preparation method according to claim 1, characterized in that, The flavor enzyme described in step (1) consists of bromelain and papain, with a mass ratio of 1:0.5-1.

6. The preparation method according to claim 1, characterized in that, The filtration in step (2) involves first filtering with a microfiltration membrane with a pore size of 0.1-0.5 µm to obtain a filtrate, and then treating the filtrate with a 5000-10000 Dalton ultrafiltration membrane.

7. The preparation method according to claim 1, characterized in that, The concentration temperature in step (3) is 40-60℃, and the concentration is carried out until the mass fraction of solid content is 10-20%.

8. The preparation method according to claim 1, characterized in that, The drying process described in step (3) is spray drying, and the inlet temperature of the spray drying process is 160-200℃.

9. Collagen tripeptide powder prepared by any one of claims 1-8.

10. The use of the collagen tripeptide powder prepared by any one of the preparation methods of claims 1-8 in the preparation of food, health products, pharmaceuticals or cosmetics.

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