Collagen active pentapeptide capable of promoting synthesis of hyaluronic acid, type I collagen and type III collagen as well as preparation method and functional product of collagen active pentapeptide

Abstract

The invention relates to the technical field of deep processing of collagen, in particular to collagen active pentapeptide capable of promoting synthesis of hyaluronic acid, I-type collagen and III-type collagen, a preparation method of the collagen active pentapeptide and a functional product of the collagen active pentapeptide. The preparation method comprises the following steps: mixing fish scales with water, and carrying out softening pretreatment at a high temperature; after pretreatment and pulping, papain and bacillus subtilis neutral protease are sequentially added for hydrolysis twice; and filtering and drying to obtain powdery collagen peptide, namely the collagen active pentapeptide. In the prepared collagen peptide, six collagen active pentapeptides capable of promoting the synthesis of hyaluronic acid, type I collagen and type III collagen can be selected by adopting a traditional separation and purification and bioinformatics combined technology, and the amino acid sequences of the collagen active pentapeptides are as follows: Ser-Asp-Gly-IIe-Phe (SDGIF), Ala-Gly-Phe-Gly-IIe (AGFGI), Ala-Phe-Gly-Leu-Ala (AFGLA), IIe-Ser-Pro-Pro-Arg (ISPPR), Ala-Gly-Leu-Gly-Leu (AGLGL) and Thr-Pro-IIe-Gln-Phe (TPIQF).

Description

Technical Field

[0001] This application relates to the technical field of deep processing of collagen, and particularly relates to a collagen active pentapeptide that promotes the synthesis of hyaluronic acid, type I and type III collagen, its preparation method, and functional products. Background Art

[0002] Collagen generally exists in the tendons, ligaments, cartilage, skin and some connective tissues of mammals and is a class of functional proteins with stable physical and chemical properties. Currently, collagen is mostly prepared industrially by extracting from the skin, bones and other parts of terrestrial mammals such as cows and pigs. Collagen peptides are proteins with a smaller molecular weight than collagen, which are beneficial for the digestion and absorption in the small intestine of the human body. The special physical and chemical properties and biological structure of collagen endow it with high nutritional characteristics and processing performance. Therefore, research on collagen is being favored by more and more researchers.

[0003] Currently, the research on collagen peptides mainly focuses on their functions, such as antioxidant, blood pressure lowering, delaying skin aging, maintaining skin elasticity and other functions. At the same time, collagen has significant low immunogenicity, biocompatibility and biodegradability, is easily absorbed in organisms, and has characteristics such as strong hydrophilicity, good non-toxic safety, etc. Therefore, it is widely used in industrial fields such as food, cosmetics, and medicine.

[0004] In recent years, using the directed enzymatic hydrolysis technology to hydrolyze collagen to obtain collagen peptides with characteristic fragments has become a research hotspot. Collagen peptides with characteristic fragments can significantly improve a certain aspect of function. For example: The Chinese invention patent application with the publication number CN116904540A discloses a process for extracting high-content collagen dipeptides and tripeptides from raw materials rich in collagen, including the following steps: controlling enzymatic hydrolysis with specific endopeptidases, exopeptidases and collagenase, inactivating the enzyme, then adsorbing and removing fishy smell with activated carbon, concentrating and spray drying for preparation. This invention can obtain products with more than 35% collagen tripeptides, more than 0.2% dipeptide Pro-Hyp, more than 0.15% dipeptide Hyp-Gly, more than 2.2% tripeptide Gly-Pro-Hyp, and more than 1.6% tripeptide Gly-Pro-Ala. The Chinese invention patent application with the publication number CN116769861A discloses a process for obtaining collagen tripeptides by enzymatically hydrolyzing fish skin with a compound protease. The collagen tripeptides obtained by this process have the characteristic of high GPH (Gly-Pro-Hyp) content, and the glycine GPH content therein can reach 3.6%.

[0005] In the future, collagen peptides with functional fragments will be widely used in cosmetics, medicine, beauty and other fields. Preparing collagen peptides with multiple active functions from fish scales has important theoretical value and practical significance. However, there is little research on how to obtain collagen peptides with functional fragments from fish scales. At present, there is no report on how to obtain collagen peptides with functional fragments that can promote the synthesis of hyaluronic acid, type I and type III collagen from fish scales, and this is exactly what technicians are committed to achieving. Summary of the Invention

[0006] To solve the deficiencies of the prior art mentioned in the above background art, the present application provides a preparation method of a collagen active pentapeptide that can promote the synthesis of hyaluronic acid, type I and type III collagen. The technical solution is as follows: The preparation method of the collagen active pentapeptide includes the following steps: Pretreatment: Mix fish scales and water evenly and heat to soften, then perform pulping treatment to obtain a pretreatment solution; Hydrolysis: Sequentially add papain and Bacillus subtilis neutral protease to the pretreatment solution for two-step hydrolysis, and then perform enzyme inactivation treatment to obtain a hydrolysis solution; Separation: Perform solid-liquid separation on the hydrolysis solution to obtain a filtrate containing collagen peptides; Drying: The filtrate is sequentially subjected to filtration to retain the filtrate and drying treatment to obtain the collagen active pentapeptide.

[0007] In some embodiments, during the pretreatment process, the mass ratio of the fish scales to the water is 1:(10-20); the temperature of the pretreatment is 90°C-95°C, and the softening time is 1-1.5 h.

[0008] In some embodiments, the addition amount of papain is 0.5%-1.0% of the mass of the fish scales; the addition amount of Bacillus subtilis neutral protease is 2.0%-2.5% of the mass of the fish scales.

[0009] In some embodiments, add papain to the pretreatment solution for the first hydrolysis. The temperature of the first hydrolysis is 50°C-55°C, the pH of the reaction system is 6.5-7.0, and the hydrolysis time is 1-3 h; then inactivate the enzyme at 85°C-90°C for 15-20 min to obtain a first hydrolysis solution.

[0010] In some embodiments, add Bacillus subtilis neutral protease to the first hydrolysis solution for the second hydrolysis. The temperature of the second hydrolysis is 50°C-55°C, the pH of the reaction system is 6.5-7.0, and the hydrolysis time is 1-3 h; then inactivate the enzyme at 85°C-90°C for 15-20 min to obtain a second hydrolysis solution.

[0011] In some embodiments, the hydrolyzate obtained from the two - stage hydrolysis is filtered for solid - liquid separation to obtain a filtrate containing collagen peptides.

[0012] In some embodiments, the filtrate is ultra - filtered to retain the filtrate containing collagen peptides with a molecular weight less than 5000 Da, and then dried to obtain the collagen active pentapeptide.

[0013] In some embodiments, spray - drying treatment is adopted to obtain the powdery collagen active pentapeptide.

[0014] This application also provides a collagen active pentapeptide, which is prepared by the preparation method of the collagen active pentapeptide as described above.

[0015] This application also provides a functional product, whose components include the collagen active pentapeptide prepared by the preparation method of the collagen active pentapeptide as described above; the functional product has at least one of the following functions: (1) Promote hyaluronic acid synthesis; (2) Promote type - I collagen synthesis; (3) Promote type - III collagen synthesis.

[0016] Compared with the prior art, this application has the following beneficial effects: The preparation method provided by this application prepares a collagen active pentapeptide with the functions of promoting hyaluronic acid synthesis, promoting type - I collagen synthesis, and promoting type - III collagen synthesis through specific extraction and purification of the collagen in fish scales. The collagen active pentapeptide with the above - mentioned functional effects can be used as a functional factor in functional products.

[0017] The method provided by this application can prepare the required polypeptide by combining simple operations such as heating and softening, enzymatic hydrolysis, filtration, and drying. It has low requirements for equipment, simple process and easy operation, and is convenient for large - scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the drawings. Obviously, the described embodiments are some, but not all, of the embodiments of this application. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0019] Figure 1 It is a qRT - PCR analysis result graph of the collagen peptide samples prepared for the examples and the comparative examples on the Col Ⅰ gene in HSF cells; Figure 2qRT-PCR analysis result graphs of collagen peptide samples prepared in the examples and comparative examples for Col Ⅲ gene in HSF cells; Figure 3 qRT-PCR analysis result graphs of collagen peptide samples prepared in the examples and comparative examples for HAS2 gene in HSF cells; Figure 4 Docking result graph of SDGIF and hyaluronidase; Figure 5 Docking result graph of SDGIF and MMP-1; Figure 6 Docking result graph of SDGIF and MMP-3; Figure 7 Docking result graph of AGFGI and hyaluronidase; Figure 8 Docking result graph of AGFGI and MMP-1; Figure 9 Docking result graph of AGFGI and MMP-3; Figure 10 Docking result graph of AFGLA and hyaluronidase; Figure 11 Docking result graph of AFGLA and MMP-1; Figure 12 Docking result graph of AFGLA and MMP-3. Detailed implementation manners

[0020] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application are described clearly and completely below. Apparently, the described embodiments are some but not all of the embodiments of the present application; the technical features designed in different implementation manners of the present application described below can be combined with each other as long as they do not conflict with each other; based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0021] In the description of the present application, it should be noted that all terms (including technical terms and scientific terms) used in the present application have the same meanings as those commonly understood by those of ordinary skill in the art to which the present application belongs, and should not be construed as a limitation to the present application; it should be further understood that the terms used in the present application should be understood as having meanings consistent with their meanings in the context of this specification and the relevant art, and should not be understood in an idealized or overly formal sense, unless otherwise clearly defined in the present application.

[0022] This application provides an operation example of a preparation method of a collagen active pentapeptide with the promotion of hyaluronic acid, type I and type III collagen synthesis. The specific steps are as follows: Step 1 (1) Pretreatment: Mix fish scales and water evenly and heat to soften, then perform pulping treatment to obtain a pretreatment solution; Among them, the mass ratio of the fish scales to pure water is 1:(10 - 20); the temperature of the pretreatment is 90°C - 95°C, and the softening time is 1 - 1.5 h; Step 2 Add papain to the pretreatment solution, adjust the system pH to 6.5 - 7.0, hydrolyze at 50°C - 55°C for 1 - 3 h, and then perform enzyme inactivation treatment at 85°C - 90°C for 15 - 20 min to obtain a first hydrolysate; among them, the addition amount of papain is 0.5% - 1.0% of the mass of the fish scales; Step 3 Add Bacillus subtilis neutral protease to the first hydrolysate, adjust the system pH to 6.5 - 7.0, hydrolyze at 50°C - 55°C for 1 - 3 h, and then perform enzyme inactivation treatment at 85°C - 90°C for 15 - 20 min to obtain a second hydrolysate; among them, the addition amount of Bacillus subtilis neutral protease is 2.0% - 2.5% of the mass of the fish scales; Step 4 Cool the second hydrolysate to room temperature and filter to obtain a filtrate containing collagen peptides. After filtration, ultrafiltration is performed to obtain a filtrate containing collagen peptides with a molecular weight lower than 5000 Da, and then spray drying treatment is performed to obtain collagen pentapeptide.

[0023] Step 6 Perform activity detection and cell experiments on the collagen peptides.

[0024] This application provides the following examples and comparative examples The enzyme activity of the Bacillus licheniformis alkaline protease, papain, Bacillus subtilis neutral protease, aminopeptidase, and ficin used in the examples and comparative examples is 200,000 U / g, 500,000 U / g, 200,000 U / g, 150,000 U / g, and 100,000 U / g, respectively.

[0025] Among them, the above enzyme activity has a different meaning from the unit U / g of the enzyme addition amount converted according to the protein mass in each g of fish scales. The meaning of the X U / g enzyme activity unit here is: the enzyme activity of each g of enzyme is X U. The unit of the enzyme addition amount converted according to the raw material protein described in this article is X U / g, which means that for 1 g of the raw material protein mass, the required enzyme activity of the added enzyme is X U.

[0026] Example 1 1. Take 500 g of fish scales (protein content 100%) and mix them evenly with 5000 g of pure water. Heat the mixture to 90 °C, soften for 1 h, and then put it into a colloid mill to make a homogeneous slurry; 2. Cool to 55 °C, adjust the pH to 7.0 with sodium hydroxide, add 0.6% of papain based on the weight of the fish scales and hydrolyze for 2 h, then heat to 90 °C to inactivate the enzyme for 15 min; 3. After cooling to 55 °C, adjust the pH to 6.5, add 2.25% of Bacillus subtilis neutral protease based on the weight of the fish scales and hydrolyze for 2 h, then heat to 85 °C to inactivate the enzyme for 20 min; 4. Obtain a filtrate containing collagen peptides by filtration, and then perform ultrafiltration to obtain a collagen peptide solution with a molecular weight less than 5000 Da; 5. Obtain collagen peptide powder by spray drying.

[0027] Example 2 1. Take 500 g of fish scales (protein content 100%) and mix them evenly with 7500 g of pure water. Heat the mixture to 90 °C, soften for 1.5 h, and then put it into a colloid mill to make a homogeneous slurry; 2. Cool to 55 °C, adjust the pH to 7.0 with sodium hydroxide, add 0.5% of papain based on the weight of the fish scales and hydrolyze for 1 h, then heat to 85 °C to inactivate the enzyme for 20 min; 3. After cooling to 55 °C, adjust the pH to 6.5, add 2.5% of Bacillus subtilis neutral protease based on the weight of the fish scales and hydrolyze for 3 h, then heat to 90 °C to inactivate the enzyme for 15 min; 4. Obtain a filtrate containing collagen peptides by filtration, and then perform ultrafiltration to obtain a collagen peptide solution with a molecular weight less than 5000 Da; 5. Obtain collagen peptide powder by spray drying.

[0028] Example 3 1. Take 500 g of fish scales (protein content 100%) and mix them evenly with 10000 g of pure water. Heat the mixture to 95 °C, soften for 1 h, and then put it into a colloid mill to make a homogeneous slurry; 2. Cool to 55 °C, adjust the pH to 6.5 with sodium hydroxide, add 1.0% of papain based on the weight of the fish scales and hydrolyze for 3 h, then heat to 90 °C to inactivate the enzyme for 15 min; 3. After cooling to 55 °C, adjust the pH to 6.0 with hydrochloric acid, add 2.0% of Bacillus subtilis neutral protease based on the weight of the fish scales and hydrolyze for 1 h, then heat to 85 °C to inactivate the enzyme for 20 min; 4. Obtain a filtrate containing collagen peptides by filtration, and then perform ultrafiltration to obtain a collagen peptide solution with a molecular weight less than 5000 Da; 5. Obtain collagen peptide powder by spray drying.

[0029] Comparative Example 1 Change the softening time in Step 1 to 0.5 h, and the remaining operations and processes are the same as those in Example 1.

[0030] Comparative Example 2 Change the softening temperature in Step 1 to 85 °C, and the remaining operations and processes are the same as those in Example 1.

[0031] Comparative Example 3 (During the hydrolysis process, Bacillus licheniformis alkaline protease and Bacillus subtilis neutral protease are used) The difference from Example 1 is only that: replace the papain in Step 2 with Bacillus licheniformis alkaline protease. In the first hydrolysis step, the enzymatic hydrolysis pH is 8.5, and the addition amount of Bacillus licheniformis alkaline protease is 7.5 g.

[0032] In Example 1, the addition amount of papain is 3 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of papain is 3000 U / g. The specific calculation process is as follows: The enzyme activity of 3 g of papain is: 500,000 U / g × 3 g = 1,500,000 U. Converted to the enzyme addition amount calculated based on the protein mass per g of fish scales, it is: 1,500,000 U / (500 g × 100%) g = 3000 U / g (the denominator g in the unit represents the protein mass per g of fish scales); Similarly, the addition amount of Bacillus subtilis neutral protease is 11.25 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of Bacillus subtilis neutral protease is 4500 U / g.

[0033] Therefore, for the 7.5 g of Bacillus licheniformis alkaline protease in this comparative example, calculated based on the protein mass in fish scales, the enzyme addition amount of Bacillus licheniformis alkaline protease is 3000 U / g (200,000 U / g × 7.5 g / (500 g × 100%)).

[0034] Comparative Example 4 (During the hydrolysis process, Bacillus licheniformis alkaline protease and papain are used) The difference from Example 1 is only that: change the Bacillus subtilis neutral protease in Step 3 to Bacillus licheniformis alkaline protease, with an addition amount of 11.25 g, the enzymatic hydrolysis pH is 8.5, and the remaining operations and processes are the same as those in Example 1.

[0035] In Example 1, the addition amount of papain is 3 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of papain is 3000 U / g. The specific calculation process is as follows: The enzyme activity of 3 g of papain is: 500,000 U / g * 3 = 1,500,000 U. The enzyme addition amount calculated based on the protein mass in each gram of fish scales is: 1,500,000 U / (500 g * 100%) g = 3000 U / g; The addition amount of Bacillus subtilis neutral protease is 11.25 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of Bacillus subtilis neutral protease is 4500 U / g; Therefore, for the 11.25 g of Bacillus licheniformis alkaline protease in this comparative example, calculated based on the protein mass in fish scales, the enzyme addition amount of Bacillus licheniformis alkaline protease is 4500 U / g.

[0036] Comparative Example 5 (using Bacillus licheniformis alkaline protease and aminopeptidase during hydrolysis) The difference from Example 1 is only that: in Step 2, papain is replaced with Bacillus licheniformis alkaline protease, with an addition amount of 7.5 g, the enzymatic hydrolysis pH is 8.5, in Step 3, Bacillus subtilis neutral protease is replaced with aminopeptidase, and the addition amount is changed to 15 g, and the rest of the operations and processes are the same as those in Example 1.

[0037] In Example 1, the addition amount of papain is 3 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of papain is 3000 U / g. The specific calculation process is as follows: The enzyme activity of 3 g of papain is: 500,000 U / g * 3 = 1,500,000 U. The enzyme addition amount calculated based on the protein mass in each gram of fish scales is: 1,500,000 U / (500 g * 100%) g = 3000 U / g; The addition amount of Bacillus subtilis neutral protease is 11.25 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of Bacillus subtilis neutral protease is 4500 U / g; Therefore, for the 7.5 g of Bacillus licheniformis alkaline protease in this comparative example, calculated based on the protein mass in fish scales, the enzyme addition amount of Bacillus licheniformis alkaline protease is 3000 U / g, and for the 15 g of aminopeptidase, calculated based on the protein mass in fish scales, the enzyme addition amount of aminopeptidase is 4500 U / g, which is equal to the total amount used of papain and Bacillus subtilis neutral protease in Example 1.

[0038] Comparative Example 6 (using Bacillus licheniformis alkaline protease and ficin during hydrolysis) The difference from Example 1 is only that: in Step 2, papain is replaced with Bacillus licheniformis alkaline protease, with an addition amount of 7.5 g, the enzymatic hydrolysis pH is 8.5, in Step 3, Bacillus subtilis neutral protease is replaced with ficin, and the addition amount is changed to 22.5 g, the temperature is raised to 65 °C, and the enzyme inactivation is at 100 °C for 20 min, and the rest of the operations and processes are the same as those in Example 1.

[0039] In Example 1, the addition amount of papain is 3 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of papain is 3000 U / g. The specific calculation process is as follows: The enzyme activity of 3 g of papain is: 500,000 U / g * 3 g = 1,500,000 U. Converting to the enzyme addition amount per g of protein mass in fish scales is: 1,500,000 U / (500 g * 100%) g = 3000 U / g; The addition amount of Bacillus subtilis neutral protease is 11.25 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of Bacillus subtilis neutral protease is 4500 U / g; Therefore, for the 7.5 g of Bacillus licheniformis alkaline protease in this comparative example, calculated based on the protein mass in fish scales, the enzyme addition amount of Bacillus licheniformis alkaline protease is 3000 U / g, and for the 22.5 g of ficin, calculated based on the protein mass in fish scales, the enzyme addition amount of ficin is 4500 U / g, which is equal to the total amount used of papain and Bacillus subtilis neutral protease in Example 1.

[0040] Comparative Example 7 (papain is used in the hydrolysis process) The difference from Example 1 is only that: the addition amount of papain in Step 2 is changed to 7.5 g, and Step 3 is removed.

[0041] In Example 1, the addition amount of papain is 3 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of papain is 3000 U / g. The specific calculation process is as follows: The enzyme activity of 3 g of papain is: 500,000 U / g * 3 g = 1,500,000 U. Converting to the enzyme addition amount per g of protein mass in fish scales is: 1,500,000 U / (500 g * 100%) g = 3000 U / g (the denominator g in the unit represents the protein mass per g of fish scales); Similarly, the addition amount of Bacillus subtilis neutral protease is 11.25 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of Bacillus subtilis neutral protease is 4500 U / g.

[0042] Therefore, for the 7.5 g of papain in this comparative example, calculated based on the protein mass in fish scales, the enzyme addition amount of papain is 7500 U / g (500,000 U / g * 7.5 g / (500 g * 100%)).

[0043] Comparative Example 8 (Bacillus subtilis neutral protease is used in the hydrolysis process) The difference from Example 1 is only that: the addition amount of Bacillus subtilis neutral protease in Step 3 is changed to 18.75 g, and Step 2 is removed.

[0044] In Example 1, the addition amount of papain is 3 g. Calculated based on the protein mass in fish scales, the enzyme addition amount of papain is 3000 U / g. The specific calculation process is as follows: The enzyme activity of 3 g of papain is: 500,000 U / g × 3 g = 1,500,000 U. The enzyme addition amount calculated based on the protein content in each gram of fish scales is: 1,500,000 U / (500 g × 100%) g = 3000 U / g (the denominator g in the unit represents the protein content in each gram of fish scales); Similarly, the addition amount of Bacillus subtilis neutral protease is 11.25 g, and based on the protein content in fish scales, its enzyme addition amount is 4500 U / g.

[0045] Therefore, for the 18.75 g of Bacillus subtilis neutral protease in this comparative example, based on the protein content in fish scales, its enzyme addition amount is 7500 U / g (200,000 U / g × 18.75 g / (500 g × 100%)).

[0046] Performance detection: Calculate the yield of the collagen peptide powder obtained from the above examples and comparative examples: For the specific yield, see Table 1: Table 1 Sensory and yield of collagen peptides with different processes

[0047] It can be seen from the comparison with Example 1 that under the same other experimental conditions, the yield of Example 1 is higher than that of Comparative Example 1. The reason is that the softening time of Comparative Example 1 is 0.5 h, and there are still many hard fish scale pieces after beating, and there are also many fish scale fragments that are not enzymatically hydrolyzed during filtration. Therefore, the softening time should be at least 1 h; although the softening time of Comparative Example 2 is also 1 h, since the softening temperature of 85 °C is lower than 90 °C of Example 1, its yield has also decreased by about 10%. Therefore, the softening temperature of fish scales should not be lower than 90 °C. In addition, the yields of Comparative Example 7 and Comparative Example 8 are also relatively low. At the same time, the collagen peptide obtained from Comparative Example 7 has a bitter taste and is difficult to apply.

[0048] The collagen active pentapeptide prepared in the example has a bland taste, no peculiar smell, and good flavor, and can be applied to food.

[0049] Conduct activity detection on the collagen peptides prepared from the above examples and comparative examples: 1. Hyaluronidase inhibitory activity of collagen peptide Prepare a 2% concentration solution (the solvent is water) of the collagen peptides prepared from the examples and comparative examples, and detect their hyaluronidase inhibition rates. The test results are shown in Table 1 (since Comparative Example 1, Comparative Example 2, Comparative Example 7, and Comparative Example 8 have problems in yield and taste and are difficult to apply, they are not detected here).

[0050] The detection method of the hyaluronidase inhibition rate of collagen peptide refers to "Carboxymethylation and acetylation modification of polysaccharides from Camellia oleifera seed meal and their inhibitory effects on hyaluronidase" and "Improved CTAB turbidimetry for the efficient determination of hyaluronic acid content in fermentation broth". The specific results are shown in Table 2: Table 2 Hyaluronidase inhibition ability of collagen peptides with different processes

[0051] It can be seen from Table 2 that the hyaluronidase inhibition rate of the examples is significantly higher than that of each comparative example, indicating that the examples can significantly inhibit hyaluronidase.

[0052] 2. RT-PCR detection of the gene expression levels of ColⅠ (human type I collagen, also known as type I collagen), ColⅢ (human type III collagen, also known as type III collagen), and HAS2 (hyaluronic acid synthase 2) in HSF cells (fibroblasts) The CCK8 method (Cell Counting Kit-8 cell counting reagent method) was used to determine cell viability. The results were analyzed by significance analysis, and the highest non-toxic concentration of 800 ug / mL was selected to add drugs to the cells.

[0053] Collect HSF cells and inoculate them in 24-well plates at a density of 2×10 4 cells / mL, and place them in an incubator for 12 h. When the cells grow to 70% - 75%, add the sample of Example 1 and the samples of Comparative Examples 3 - 6 with the cell safety dose concentration screened by CCK-8. Another blank control group (control group) was set up, with 3 parallel replicates in each group, and cultured for 24 h. Then, the total cell RNA was extracted according to the method of Simply P total RNA extraction kit (BioFlux). Take 2 μL of sterile and enzyme-free water and drop it on the concentration measurement plate (Nanodrop one) to detect the RNA concentration and purity. A 20 μL qRT-PCR reaction system was established, and the PCR amplification conditions were: Pre-denaturation at 95℃ for 30 s; denaturation at 95℃ for 15 s, annealing at 54℃ for 30 s, extension at 72℃ for 45 s, and the reaction cycle was 40 times. The reaction conditions for the melting curve were: 95℃ for 30 s, 60℃ for 1 min, and the fluorescence signal was collected once every 5℃ increase. The primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd. Using GAPDH as an internal reference, the primer sequence information is as follows: GAPDH, 5'-CTATAAATTGAGCCCGCAGC-3' (F) and 5'-GACCAAATCCGTTGACTCCG-3' (R); ColⅠ, 5'-GGCAAAGAAGGCGGCAAAGG-3' (F) and 5'-GGAGCACCAGCAGGACCATC-3' (R); ColⅢ, 5'-CCAAAGGGTGACAAGGGTGAAC-3' (F) and 5'-CAGGAGGACCAATAGGACCAGTAG-3' (R); HAS2, 5'-GGGGCACATCAGGAAGGAAAACC-3' (F) and 5'-CACCACCCAGGAGGATTGTAAACC-3' (R).

[0054] It should be noted that the above primers are all existing primers.

[0055] The qRT-PCR analysis results of the Col I gene in HSF cells, the qRT-PCR analysis results of the Col III gene in HSF cells, and the qRT-PCR analysis results of the HAS2 gene in HSF cells for the collagen peptide samples prepared in the examples and comparative examples are respectively as Figures 1-3 shown.

[0056] As Figure 1 , Figure 2 and Figure 3 shown, on the premise of ensuring the stable growth state of cells and not affecting the proliferation ability, compared with the comparative example, Example 1 can significantly increase the expression levels of Col I mRNA and Col III mRNA in HSF cells, promote the synthesis of Col I and Col III, and ultimately promote the synthesis of collagen in the dermal layer of the skin, indicating that the collagen peptide of Example 1 can regulate the metabolic process of collagen and maintain the structural stability of collagen fibers and the extracellular matrix. At the same time, Example 1 can significantly increase the expression level of HAS2mRNA in HSF cells, promote the synthesis and secretion of hyaluronic acid in the skin, and increase the firmness and moisture of the skin.

[0057] Peptide segment screening of collagen peptide 3.1 Pretreatment method 3.1.1 Reductive alkylation 1) Weigh 10.4 mg and 11.3 mg of the samples respectively, dissolve them thoroughly with 100 μL of water, and conduct subsequent experiments; 2) Accurately pipette 1 μL of 1M DTT (dithiothreitol) solution into the samples to make the final concentration of DTT 10 mmol / L, and reduce at 56°C in a water bath for 1 h; 3) Use a pipette to accurately transfer 2 μL of 1M IAM (2-iodoacetamide) solution into the sample to make the final concentration of IAM 20 mmol / L, and react at room temperature in the dark for 40 min; 4) Use a pipette to accurately transfer 1 μL of 1M DTT solution into the sample to make the final concentration of DTT 10 mmol / L to neutralize the unreacted IAM.

[0058] 3.1.2 C18 Desalting (Stage-Tip) 1) Desalt using a C18 stage-tip and dry under vacuum at 45 °C.

[0059] 3.2 Instrumentation Inject the processed sample into a liquid chromatography-mass spectrometry instrument for detection. The instrument conditions are as follows: 3.2.1 Liquid Chromatography Conditions 1) Pre-column: 150 μm i.d. × 50 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm; Analytical column: 150 μm i.d. × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm; 2) Mobile phase A: 0.1% FA (formic acid); 3) Mobile phase B: 0.1% FA, 80% ACN (acetonitrile); 4) Flow rate: 600 nL / min; 5) Analysis time for each component: 66 min; 6) Specific chromatographic conditions are shown in Table 3: Table 3

[0060] 3.2.2 Mass Spectrometry Conditions Primary mass spectrometry parameters: 1) Resolution: 70,000; 2) AGC target: 3e6; 3) Maximum IT: 100 ms; 4) Scan range: 100 to 1500 m / z; Secondary mass spectrometry parameters: 1) Resolution: 17,500; 2) AGC target: 1e5; 3) Maximum IT: 50 ms; 4) TopN: 20; 5) NCE / stepped NCE: 28; Obtain the original raw data after mass spectrometry acquisition.

[0061] 3.3 Search Library Conditions The original mass spectrometry files are used to search the target protein database with the software, and the search parameters are as follows: 1) Fixed modifications: Carbamidomethyl (C).

[0062] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term).

[0063] 3) Enzyme: Non specific.

[0064] 4) Analysis database: uniprotkb_Acipenser (genus)_2024_11_13; uniprotkb_Oreochromis (genus)_2024_12_05.

[0065] 5) Peptide Mass Tolerance: 20 ppm; 6) Fragment Mass Tolerance: 0.02 Da.

[0066] The raw files collected by mass spectrometry are searched against the Byonic database with the software, and the peptide list obtained is shown below. The following table shows 9 pentapeptide segments sorted by abundance after screening by abundance conditions in Example 1, as shown in Table 4 specifically: Table 4 Information of 9 Peptide Segments Obtained after Screening

[0067] Note: Peptide<ProteinMetrics Confidential>: Amino acid sequence of the identified peptide segment; Observed m / z: Mass-to-charge ratio actually detected for the peptide segment; z: Charge number carried by the peptide segment; Mass error(ppm): Molecular weight error; Score: Peptide segment score; Scan Time: Retention time of the peptide segment; Intensity: Abundance of the peptide segment; After retrieving from the polypeptide database BIOPEP (http: / / bis.zju.edu.cn / biopepdbr / index.php), all 9 peptide segments are novel and have not been reported before. They are predicted to be non-toxic by the peptide toxicity prediction website (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php). The molecular docking software was used to perform molecular docking of the above 9 fragments with hyaluronidase against MMP-1, MMP-3, and the hyaluronidase receptor. The three-dimensional structure diagrams of MMP-1 (PDB code: 966c), MMP-3 (PDB code: 2JT6), and hyaluronidase (PDB code: 2Pe4) were downloaded from the PDB database (http: / / www.rcsb.org / ). The docking binding energy data of the specific 9 peptide segments with different receptors are shown in Table 5 as follows: Table 5 Binding Energies of Peptide Segments Docked with Receptor Proteins (MMP-1, MMP-3, Hyaluronidase Receptor 2Pe4)

[0068] Note: In Table 5, the unit is kcal / mol.

[0069] Among them, hyaluronidase (2Pe4) is a type of enzyme that can decompose hyaluronic acid, and MMP-1 and MMP-3 are the main enzymes for degrading type I collagen (Col I) and type III collagen (Col III). As shown in the results of Table 5, except that QIGLR could not be docked with MMP-3, the remaining peptides have certain binding energies with the three receptor proteins of hyaluronidase 2Pe4, MMP-1, and MMP-3. At the same time, by inputting the 9 peptide segments into the scoring website ( http: / / distilldeep.ucd.ie / PeptideRanker / ) for scoring, the scores of six peptide segments, namely SDGIF, AGFGI, AFGLA, ISPPR, AGLGL, and TPIQF, are > 0.5 (the higher the score, the higher the potential biological activity). Therefore, the six peptide segments SDGIF, AGFGI, AFGLA, ISPPR, AGLGL, and TPIQF are effective active fragments of the collagen-active pentapeptides that promote the synthesis of hyaluronic acid, type I, and type III collagen.

[0070] Figures 4-6 They respectively represent the 2D diagrams of the molecular docking of SDGIF with the three receptor proteins of MMP-1, MMP-3, and hyaluronidase, Figures 7-9 They respectively represent the 2D diagrams of the molecular docking of AGFGI with the three receptor proteins of MMP-1, MMP-3, and hyaluronidase, Figures 10-12Respectively shown are the 2D diagrams of the docking of AFGLA with three receptor protein molecules, namely MMP-1, MMP-3, and hyaluronidase.

[0071] As Figure 4 shown, the interactions between SDGIF and hyaluronidase are through carbon-hydrogen bonds, van der Waals forces, and covalent bonds; as Figure 5 shown, the interactions between SDGIF and MMP-1 are mainly through conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, and van der Waals forces; as Figure 6 shown, the interactions between SDGIF and MMP-3 are mainly through conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, and van der Waals forces. As Figure 7 shown, the interactions between AGFGI and hyaluronidase are through carbon-hydrogen bonds, van der Waals forces, and covalent bonds; as Figure 8 shown, the interactions between AGFGI and MMP-1 are mainly through conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, and van der Waals forces; as Figure 9 shown, the interactions between AGFGI and MMP-3 are mainly through conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, and van der Waals forces. As Figure 10 shown, the interactions between AFGLA and hyaluronidase are through carbon-hydrogen bonds, van der Waals forces, and covalent bonds; as Figure 11 shown, the interactions between AFGLA and MMP-1 are mainly through conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, van der Waals forces, and salt bridges,; as Figure 12 shown, the interactions between AFGLA and MMP-3 are mainly through conventional hydrogen bonds, carbon-hydrogen bonds, attractive charges, and van der Waals forces, etc.

[0072] In summary, it can be known that: Hyaluronidase is a class of enzymes that can decompose hyaluronic acid. MMP-1 and MMP-3 are the main enzymes for degrading type I collagen (Col I) and type III collagen (Col III). Since multiple peptide segments in the collagen peptide prepared in the embodiments of the present application have a certain binding energy with three receptor proteins, namely hyaluronidase 2Pe4, MMP-1, and MMP-3, the catabolism of collagen can be effectively inhibited, the synthesis of type I collagen (Col I) and type III collagen (Col III) can be promoted. Similarly, it can inhibit the activity of hyaluronidase and promote the synthesis of hyaluronic acid.

[0073] In summary, the present application has the following beneficial effects: The present application prepares a collagen active pentapeptide with the functions of promoting the synthesis of hyaluronic acid, promoting the synthesis of type I collagen, and promoting the synthesis of type III collagen through specific extraction and purification of the collagen in fish scales.

[0074] The collagen active pentapeptide can be used as a functional factor in products with obvious effects such as beauty and skin care, playing roles such as increasing the firmness and tenderness of the skin. For example, it can be applied to oral active peptide liquid products or external skin care products, etc. The solution of this application provides a theoretical basis and practical operation plan for the deep processing and industrialization of collagen.

[0075] The method provided by this application can obtain the required polypeptide by combining simple operations such as heating and softening, enzymatic hydrolysis, filtration, and drying. It has low requirements for equipment, simple process and easy operation, and is convenient for large-scale industrial production.

[0076] It should be noted that: (1)Definition: In this article, "~" is used to represent the numerical range, and both endpoint values are included within the represented range.

[0077] The full name of the term "Da" used in this article is Dalton, which is a commonly used unit of molecular weight.

[0078] The "ultrafiltration" and "spray drying" mentioned in this article are the names of conventional processing steps in the art, and their names can accurately express their processing processes, so they will not be elaborated here.

[0079] (2)Raw materials used in the implementation: The enzymes such as Bacillus licheniformis alkaline protease, papain, Bacillus subtilis neutral protease, ficin, and aminopeptidase used are all existing commercially available enzymes, which can be purchased and obtained by those skilled in the art.

[0080] (3)Application of the collagen active pentapeptide: The collagen active pentapeptide has functions such as promoting hyaluronic acid synthesis, promoting type I collagen synthesis, and promoting type III collagen synthesis. Based on the above characteristics and functions, the collagen active pentapeptide can be applied to functional products with obvious effects such as beauty and skin care (functional products can be foods, health products, cosmetics, skin care products, bath products, cleaning products, etc., including any substances that provide preventive and / or other beneficial effects).

[0081] According to the above concept, due to the functions of the collagen active pentapeptide in promoting hyaluronic acid synthesis, promoting type I collagen synthesis, and promoting type III collagen synthesis, based on the relevance of the above functional characteristics to the physiological mechanisms of humans or animals and the relevance to the occurrence and development of human diseases, the collagen active pentapeptide can also be applied to products with other obvious functions, including but not limited to obvious effects such as beauty and skin care.

[0082] In addition, the components of the functional product described in this article include the collagen active pentapeptide prepared by the preparation method of the collagen active pentapeptide, which does not limit that the functional product must be compounded with other components on the basis of containing the collagen active pentapeptide. Its components can be only the collagen pentapeptide, or the components can include the collagen pentapeptide and other ingredients (including but not limited to other peptides, excipients, etc.).

[0083] In summary, the specific parameters, some common reagents or raw materials in the above embodiments are specific embodiments or preferred embodiments under the concept of this application, rather than limitations thereto; those skilled in the art can make adaptive adjustments within the concept and protection scope of this application.

[0084] In addition, unless otherwise specified, the raw materials used can also be conventional commercially available products in the art, or prepared by conventional methods in the art; that is, the reagents and instruments used in this embodiment are not marked with information such as the manufacturer, and are all conventional products that can be obtained through market purchase.

[0085] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of this application, rather than limitations thereto; although this application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A preparation method of a collagen active pentapeptide, characterized in that, It includes the following steps: Pretreatment: Mix fish scales and water evenly and heat to soften, then perform beating treatment to obtain a pretreatment solution; Hydrolysis: Sequentially add papain and Bacillus subtilis neutral protease to the pretreatment solution for two-step hydrolysis, and then perform enzyme inactivation treatment to obtain a hydrolysis solution; Separation: Perform solid-liquid separation on the hydrolysis solution to obtain a filtrate containing collagen peptides; Drying: After the filtrate is successively subjected to filtration to intercept the filtrate and drying treatment, the collagen active pentapeptide is obtained.

2. The preparation method of the collagen active pentapeptide according to claim 1, characterized in that: In the pretreatment process, the mass ratio of the fish scales to the water is 1:(10-20); The temperature of the pretreatment is 90°C-95°C, and the softening time is 1-1.5 h.

3. The preparation method of the collagen active pentapeptide according to claim 1, characterized in that: The addition amount of papain is 0.5%-1.0% of the mass of the fish scales; The addition amount of Bacillus subtilis neutral protease is 2.0%-2.5% of the mass of the fish scales.

4. The preparation method of the collagen active pentapeptide according to claim 1, characterized in that: Add papain to the pretreatment solution for the first hydrolysis. The temperature of the first hydrolysis is 50°C-55°C, the pH of the reaction system is 6.5-7.0, and the hydrolysis time is 1-3 h; then inactivate the enzyme at 85°C-90°C for 15-20 min to obtain a first hydrolysis solution.

5. The preparation method of the collagen active pentapeptide according to claim 4, characterized in that: Add Bacillus subtilis neutral protease to the first hydrolysis solution for the second hydrolysis. The temperature of the second hydrolysis is 50°C-55°C, the pH of the reaction system is 6.5-7.0, and the hydrolysis time is 1-3 h; then inactivate the enzyme at 85°C-90°C for 15-20 min to obtain a second hydrolysis solution.

6. The preparation method of the collagen active pentapeptide according to claim 1, wherein: Filter the hydrolysis solution obtained from the two hydrolyses for solid-liquid separation to obtain a filtrate containing collagen peptides.

7. The preparation method of the collagen active pentapeptide according to claim 1, characterized in that: Ultrafilter the filtrate to intercept the filtrate containing collagen peptides with a molecular weight less than 5000 Da, and then perform drying treatment to obtain the collagen active pentapeptide.

8. The preparation method of the collagen active pentapeptide according to claim 1, characterized in that: Adopt spray drying treatment to obtain the powdery collagen active pentapeptide.

9. Collagen active pentapeptide, characterized in that: Prepared by using the preparation method of the collagen active pentapeptide according to any one of claims 1-8.

10. A functional product, characterized in that: Its components include the collagen active pentapeptide prepared by using the preparation method of the collagen active pentapeptide according to any one of claims 1-8; the functional product has at least one of the following functions: (1) Promote hyaluronic acid synthesis; (2) Promote type I collagen synthesis; (3) Promote type III collagen synthesis.

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