Grass carp skin collagen peptide and purification method and application

Abstract

The application provides a grass carp skin collagen peptide and a purification method and application, an amino acid sequence is Gly-His-Arg-Gly-Phe, and the grass carp skin collagen peptide is used for preparing health care products, cosmetics and medical beauty products. The application has the beneficial effects that: the grass carp skin collagen peptide of the application can inhibit the content of cAMP in cells, further inhibit the activity of PKA and CREB, down-regulate the expression of MITF, finally inhibit the expression of related proteins TYR, TRP-1 and TRP-2 of melanin synthesis, and inhibit the synthesis of melanin; meanwhile, the content of intracellular MDA is obviously decreased, and the grass carp skin collagen peptide can also block the synthesis and conversion of melanin by relieving the oxidative stress in cells.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation technology, and in particular relates to a grass carp skin collagen peptide, its purification method, and its application. Background Technology

[0002] Grass carp skin collagen peptide GHRGF is a product obtained by hydrolyzing grass carp skin collagen. Grass carp skin collagen is widely available and inexpensive. Fish skin collagen has advantages such as high nutritional value and high content of essential amino acids. Collagen peptides prepared from collagen can maintain or even improve the biological activity of collagen, while also having advantages such as high solubility, high absorption rate, fast effect, and high stability, making it more widely applicable.

[0003] Skin-whitening active peptides refer to polypeptides that can inhibit melanin synthesis or eliminate melanin in the skin, reducing pigmentation and thus achieving a skin-whitening effect. Their mechanisms of action mainly include inhibiting tyrosinase activity, regulating the expression of tyrosinase-related genes, and scavenging free radicals. Grass carp is an important freshwater aquaculture fish in my country, and its skin collagen peptides contain abundant bioactive components with excellent skin-whitening activity. Therefore, isolating skin-whitening active peptides from grass carp skin and studying their skin-whitening mechanism not only broadens the application of natural skin-whitening active peptides but also lays a solid foundation for the artificial synthesis of skin-whitening active peptides, demonstrating their advantages in applications in the cosmetics and food industries.

[0004] Screening for potentially active peptides from a vast amount of peptide sequence data in traditional laboratories involves synthesizing these peptides one by one and then verifying their bioactivity individually. Bioinformatics, on the other hand, is a technique that utilizes biological databases and mathematical models to screen for active peptides. It is low-cost, has a short experimental cycle, and is highly efficient. Based on machine learning methods, it typically requires first collecting and organizing protein data to build a database, then training a predictive model using the database. The trained model can then predict the activity of peptides based on their physicochemical properties and amino acid composition. By predicting the activity of peptides with known sequences using the model, molecular docking techniques and biological experiments are then used for verification, thereby screening for peptides with specific biological activities. Summary of the Invention

[0005] In view of this, the present invention aims to provide a grass carp skin collagen peptide, a purification method, and its application, in order to solve at least one technical problem in the background art.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A type of grass carp skin collagen peptide with the amino acid sequence Gly-His-Arg-Gly-Phe.

[0008] Uses of grass carp skin collagen peptides in the preparation of health products, cosmetics, and medical aesthetic products.

[0009] The use of grass carp skin collagen peptides in skin whitening products.

[0010] The purification method for the above-mentioned grass carp skin collagen peptides includes the following steps:

[0011] S1: Pre-treatment of grass carp skin;

[0012] S2: The fish skin pretreated in step S1 is swelled in acetic acid solution, pepsin is added for enzymatic hydrolysis, and after enzymatic hydrolysis, it is filtered and centrifuged. Salt is added to the supernatant while stirring, and after standing and centrifuging, the precipitate is collected, dissolved in acetic acid, dialyzed to obtain dialysate, and then freeze-dried under vacuum to obtain purified grass carp skin collagen.

[0013] S3: Prepare a collagen solution by mixing the purified grass carp skin collagen from step S2 with the buffer solution; add collagenase for hydrolysis, boil the hydrolysate, cool to room temperature, and centrifuge to obtain grass carp skin collagen hydrolysate;

[0014] Preferably, the purified collagen is prepared into a collagen solution with a mass concentration of 2.3-2.5% using buffer solution, 7% collagenase (m / m) is added, the pH is adjusted to 7.4-7.3, and hydrolyzed at 41.7℃ for 4 hours;

[0015] S4: Screen the grass carp skin collagen hydrolysate obtained in step S3 to obtain the grass carp skin collagen peptide as described in claim 1.

[0016] Preferably, active titanium with a molecular weight of less than 1000 is first screened out, and then the grass carp skin collagen peptide described in claim 1 is screened out from the isolated active peptides.

[0017] Furthermore, the pretreatment of grass carp skin in step S1 includes:

[0018] A1: Thaw the frozen grass carp skin at room temperature, wash it, scrape off any remaining fish meat, scales and bones, cut it into small pieces of the same size, and put it in an oven to dry and remove moisture to obtain completely dried grass carp skin.

[0019] A2: Cut the completely dried grass carp skin into small pieces, weigh the fish skin, soak it in n-butanol solution, change the n-butanol solution several times, rinse it clean with running water, and then treat it with NaOH solution containing salt to remove the impurities in the fish skin to obtain pre-treated grass carp skin.

[0020] Preferably, the salt in the NaOH solution is NaCl.

[0021] Further, the grass carp skin after pretreatment in step S2 is swollen in a 0.6-0.8M acetic acid solution for 10-14 h at a material-to-liquid ratio of 1:55-65 g / mL;

[0022] Preferably, the amount of pepsin added is 1900-2100 U / g;

[0023] Preferably, in step S2, the swollen pepsin and acetic acid are stirred at 28-32°C for 4-5 hours, filtered, and the filtrate is centrifuged at 3900-4100 r / min for 28-32 min, and the supernatant is retained.

[0024] Preferably, salt is slowly added to the supernatant while stirring until the concentration reaches 2.4-2.6 M. After standing for 20-24 h, the supernatant is centrifuged at 3900-4100 r / min for 29-31 min, the liquid is discarded and the precipitate is collected. The collected precipitate is redissolved in 0.4-0.6 M acetic acid and dialyzed with running water for 46-50 h. The dialysate is collected and freeze-dried under vacuum until it reaches equilibrium weight to obtain purified grass carp skin collagen.

[0025] A composition containing the above-mentioned grass carp skin collagen peptides.

[0026] The above-mentioned grass carp skin collagen peptide composition is used in the preparation of health products, cosmetics, and medical aesthetic products.

[0027] The use of the above-mentioned grass carp skin collagen peptide composition in whitening products.

[0028] A method for preparing a composition of grass carp skin collagen peptides includes the following steps:

[0029] S1: Pretreatment of grass carp skin and retrograde flow;

[0030] S2: The fish skin pretreated in step S1 is swelled in acetic acid solution, pepsin is added for enzymatic hydrolysis, and after enzymatic hydrolysis, it is filtered and centrifuged. Salt is added to the supernatant while stirring, and after standing and centrifuging, the precipitate is collected, dissolved in acetic acid, dialyzed to obtain dialysate, and then freeze-dried under vacuum to obtain purified grass carp skin collagen.

[0031] S3: Prepare a collagen solution by mixing the purified grass carp skin collagen from step S2 with a buffer solution; add collagenase for hydrolysis, boil the hydrolysate, cool to room temperature, and centrifuge to obtain grass carp skin collagen hydrolysate, which is the composition of grass carp skin collagen peptides.

[0032] Preferably, the purified collagen is prepared into a collagen solution with a mass concentration of 2.3-2.5% using buffer solution, 7% collagenase (m / m) is added, the pH is adjusted to 7.4-7.3, and hydrolyzed at 41.7℃ for 4 hours;

[0033] Preferably, the pretreatment of grass carp skin in step S1 includes:

[0034] A1: Thaw the frozen grass carp skin at room temperature, wash it, scrape off any remaining fish meat, scales and bones, cut it into small pieces of the same size, and put it in an oven to dry and remove moisture to obtain completely dried grass carp skin.

[0035] A2: Cut the completely dried grass carp skin into small pieces, weigh the fish skin, soak it in n-butanol solution, change the n-butanol solution several times, rinse it clean with running water, and then treat it with NaOH solution containing salt to remove the impurities in the fish skin to obtain pre-treated grass carp skin.

[0036] Preferably, the salt in the NaOH solution is NaCl;

[0037] Preferably, the grass carp skin after pretreatment in step S2 is swollen in a 0.6-0.8 M acetic acid solution for 10-14 h at a material-to-liquid ratio of 1:55-65 g / mL;

[0038] Preferably, the amount of pepsin added is 1900-2100 U / g;

[0039] Preferably, in step S2, the swollen pepsin and acetic acid are stirred at 28-32°C for 4-5 hours, filtered, and the filtrate is centrifuged at 3900-4100 r / min for 28-32 min, and the supernatant is retained.

[0040] Preferably, salt is slowly added to the supernatant while stirring until the concentration reaches 2.4-2.6 M. After standing for 20-24 h, the supernatant is centrifuged at 3900-4100 r / min for 29-31 min, the liquid is discarded and the precipitate is collected. The collected precipitate is redissolved in 0.4-0.6 M acetic acid and dialyzed with running water for 46-50 h. The dialysate is collected and freeze-dried under vacuum until it reaches equilibrium weight to obtain purified grass carp skin collagen.

[0041] Compared with existing technologies, the grass carp skin collagen peptide, purification method, and application described in this invention have the following advantages:

[0042] The grass carp skin collagen peptide of this application inhibits the content of cAMP in cells, thereby inhibiting the activity of PKA and CREB, downregulating the expression of MITF, and ultimately inhibiting the expression of melanin synthesis-related proteins TYR, TRP-1 and TRP-2, thus inhibiting melanin synthesis; at the same time, the intracellular MDA content is significantly reduced, and the grass carp skin collagen peptide can also block the synthesis and transformation of melanin by alleviating intracellular oxidative stress. Attached Figure Description

[0043] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0044] Figure 1 This is a schematic diagram illustrating the effect of different concentrations of GHRGF on the proliferation of B16F10 cells as described in the embodiments of the present invention;

[0045] Figure 2 This is a schematic diagram illustrating the inhibitory effect of GHRGF on melanin synthesis in B16F10 cells according to an embodiment of the present invention (A is a schematic diagram of the effect on the relative content of intracellular melanin, and B is a schematic diagram of the effect on the relative content of extracellular melanin).

[0046] Figure 3 This is a schematic diagram illustrating the effect of GHRGF on tyrosinase activity in B16F10 cells according to an embodiment of the present invention.

[0047] Figure 4 This is a schematic diagram illustrating the effect of GHRGF on MDA content in B16F10 cells according to an embodiment of the present invention;

[0048] Figure 5 This is a schematic diagram illustrating the effect of GHRGF on cAMP levels in B16F10 cells according to an embodiment of the present invention (A is a schematic diagram illustrating the effect on intracellular cAMP levels, and B is a schematic diagram illustrating the effect on extracellular cAMP levels).

[0049] Figure 6 The diagram illustrates the effects of GHRGF on the transcriptional levels of genes in the cAMP / PKA / CREB signaling pathway in B16F10 cells as described in this embodiment of the invention (A is the effect on the TYR gene in B16F10 cells, B is the effect on the TRP-1 gene in B16F10 cells, C is the effect on the TRP-2 gene in B16F10 cells, D is the effect on the MTF gene in B16F10 cells, E is the effect on the PKA gene in B16F10 cells, and F is the effect on the CREB gene in B16F10 cells).

[0050] Figure 7Schematic diagram of the effect of GHRGF on the expression levels of cAMP / PKA / CREB signaling pathway proteins in B16F10 cells (A is a schematic diagram of protein expression in B16F10 cells, B is a schematic diagram of the effect on TYR protein expression in B16F10 cells, C is a schematic diagram of the effect on TRP-1 protein expression in B16F10 cells, D is a schematic diagram of the effect on TRP-2 protein expression in B16F10 cells, E is a schematic diagram of MTF protein expression in B16F10 cells, F is a schematic diagram of the effect on PKA protein expression in B16F10 cells, and G is a schematic diagram of the effect on CREB protein expression in B16F10 cells). Detailed Implementation

[0051] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0052] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0053] A grass carp skin collagen peptide GHRGF with whitening activity, characterized in that the molecular weight of the GHRGF is 572.5 Da and the amino acid composition is Gly-His-Arg-Gly-Phe.

[0054] A method for isolating and purifying GHRGF, a collagen peptide from grass carp skin with whitening activity, includes the following steps:

[0055] (1) Thaw the frozen grass carp skin at room temperature, wash it with running tap water, scrape off any remaining fish meat, scales, and bones, cut it into 8 cm × 8 cm pieces, and then dry it in an oven to remove moisture. Cut the completely dried grass carp skin into 0.5 cm × 0.5 cm pieces, weigh about 10 g of fish skin and transfer it to a beaker, accurately recording the weight of the fish skin. Soak the fish skin in a 10% n-butanol solution for 24 h (solid-to-material ratio 1:20), changing the n-butanol solution every 8 h to remove the oil from the fish skin. Rinse it thoroughly with running water, and then treat it with a 0.1 M NaOH solution containing 3% NaCl for 6 h to remove any impurities in the fish skin.

[0056] (2) The pretreated grass carp skin was swollen in 0.7 M acetic acid solution at a material-to-liquid ratio of 1:60 (g / mL) for 12 h. Pepsin was added at a ratio of 2000 U / g, and the mixture was stirred at 30℃ for 4.5 h. The mixture was filtered through medical gauze, and the filtrate was centrifuged at 4000 r / min for 30 min, and the supernatant was collected. NaCl was slowly added to the supernatant while stirring until the concentration reached 2.5 M. The mixture was allowed to stand for 20-24 h, and then centrifuged at 4000 r / min for 30 min. The liquid was discarded and the precipitate was collected. The collected precipitate was redissolved in 0.5 M acetic acid and dialyzed continuously with running water for 48 h. The dialysate was harvested and freeze-dried under vacuum until it reached equilibrium weight to obtain purified grass carp skin collagen.

[0057] (3) The purified collagen was prepared into a collagen solution with a mass concentration of 2.4% using PBS buffer, and 7% collagenase (m / m) was added to adjust the pH to 7.5. The solution was hydrolyzed at 41.7℃ for 4 hours. After the enzymatic hydrolysis was completed, the solution was boiled in a boiling water bath for 10 minutes and then cooled to room temperature. The hydrolysate of grass carp skin collagen was centrifuged at 3000 g for 30 minutes in an ultrafiltration centrifuge tube with a molecular weight of 1 kDa.

[0058] (4) The polypeptide composition of grass carp skin collagen hydrolysate with a molecular weight of <1 kDa was analyzed by LC-MS / MS combined with Byonic software. A total of 354 polypeptides derived from grass carp collagen were identified. Using Byonic score, PeptideRanker score, content and water solubility as indicators, 8 suitable grass carp skin collagen peptides were screened out. Taking into account the amino acid composition, ADMET properties, free radical scavenging ability, tyrosinase binding energy and in vitro tyrosinase inhibitory activity, grass carp skin collagen peptide GHRGF with excellent whitening activity was screened out.

[0059] Using B16F10 mouse melanoma cells cultured in vitro as a model, the effects of the above-mentioned grass carp skin collagen peptide GHRGF on the proliferation rate, intracellular and extracellular melanin content, and intracellular tyrosinase activity of B16F10 cells were analyzed to evaluate its skin whitening activity. Based on this, qRT-PCR and Western blot techniques were used to analyze the regulatory role of GHRGF on related genes and proteins in the cAMP / PKA / CREB pathway.

[0060] Effect of GHRGF on the proliferation rate of B16F10 cells:

[0061] Logarithmic growth phase B16F10 cells were hydrolyzed with trypsin to prepare a cell suspension of 10⁵ cells / mL, and 100 μL was added to each well of a 96-well plate. After 24 h of culture, the culture medium was discarded. 100 μL of culture medium was added to the control group, and different concentrations (2000, 1000, 500, 250, 125, 62.5 μM) of GHRGF solution were added to the treatment groups. Three replicate wells were set up for each group. After incubation at 37℃ and 5% CO₂ for 48 h, the culture medium was discarded, and the cells were washed three times with PBS. 100 μL of complete culture medium containing 10% CCK-8 was added to each well, gently shaken to mix, and incubated at a constant temperature for 45 min. The absorbance value of each well was measured and recorded at a wavelength of 450 nm. Cell viability was calculated using the following formula:

[0062] ;

[0063] Among them, A 处理 It is the absorbance value of the treatment group, A 空白 This is the absorbance value of the blank group.

[0064] from Figure 1 It can be seen that, after 24 h of GHRGF treatment, compared with the control group, none of the selected concentrations had a significant toxic effect on the proliferation of B16F10 cells.

[0065] Effects of GHRGF on intracellular and extracellular melanin content in B16F10 cells:

[0066] 2 mL of cell suspension at a concentration of 10⁵ cells / mL was seeded into each well of a 6-well plate. After 24 h of culture, the culture medium was discarded, and cells were treated with 2000, 1000, 500, and 250 μM GHRGF, respectively. The blank control group was replaced with an equal volume of DMEM medium, and the positive control group was treated with 500 μM arbutin. Cells were cultured at 37°C and 5% CO₂ for 48 h. Cells were washed with 200 µL of cold PBS. 250 µL of RIPA (strong) lysis buffer containing 1% PMSF was added to each well. Lysis was performed on ice for 20 min, and the contents of the wells were transferred to centrifuge tubes using a cell scraper. Centrifugation was performed at 12000 g and 4°C for 10 min. The precipitate was collected and dissolved in 500 µL of 1 M NaOH containing 10% DMSO at 80°C for 1 h. The absorbance of each well at 450 nm was measured, and the melanin content in the cells was calculated.

[0067] Depend on Figure 2 It can be seen that, compared with the control group, the positive control arbutin and all concentrations of GHRGF showed significant inhibitory effects on the content of melanin in both the intracellular and extracellular cells of B16F10 cells, and the content of melanin in both the intracellular and extracellular cells showed similar trends. As the concentration of GHRGF increased, the content of melanin decreased, showing a significant dose-response effect.

[0068] Effects of GHRGF on intracellular tyrosinase activity in B16F10 cells:

[0069] Tyrosinase activity was measured by measuring the rate of dopachrome formation using L-DOPA as a substrate. 100 µL of the supernatant from lysis was added to each well of a 96-well plate, followed by an equal volume of 1 mg / mL L-DOPA solution. The absorbance was measured at 450 nm. The reaction mixture was incubated at 37°C for 1 h, and the absorbance at 450 nm was measured again. The difference between the two absorbance values ​​reflected tyrosinase activity. Protein concentration was determined using the Omni-Easy™ ready-to-use BCA protein quantification kit, and protein normalization was performed. Tyrosinase activity was expressed as a percentage change compared to the control.

[0070] Depend on Figure 3 It was found that, compared with the control group, the activity of tyrosinase in each treatment group was significantly reduced in a dose-dependent manner. This indicates that GHRGF can significantly inhibit the activity of tyrosinase in B16F10 cells, thus achieving a skin-whitening effect. When the concentration of GHRGF reached 2000 μM, the inhibition rate of tyrosinase activity reached 22.33%. The trend of tyrosinase activity was consistent with the trend of melanin content.

[0071] Effect of GHRGF on MDA content in B16F10 cells:

[0072] Prepare the reaction system as shown in Table 1 according to the instructions.

[0073] Table 1 Experimental system for MDA content determination

[0074] Reagent (μL) Measurement tube Blank tube MDA detection working solution 300 300 distilled water - 100 lysis supernatant 100 - Reagent 3 100 100

[0075] The mixture was boiled in a water bath for 60 min, cooled in an ice bath, and centrifuged at 10000 g for 10 min. The absorbance of each sample was measured at 532 nm and 600 nm. ΔA532 = A532 measurement - A532 blank, ΔA600 = A600 measurement - A600 blank, ΔA = ΔA532 - ΔA600. The MDA content (nmol / mg prot) was calculated based on the protein concentration.

[0076] Figure 4 The results show the MDA content differences between the different groups after GHRGF treatment. Compared with the control group, the MDA content of all treatment groups decreased significantly, except for the 250 μM concentration group, which showed no significant difference.

[0077] Effect of GHRGF on cAMP levels in B16F10 cells:

[0078] The intracellular cAMP content was determined using the cAMP ELISA Kit from Beijing Solarbio Science & Technology Co., Ltd.

[0079] Depend on Figure 5 It was found that for intracellular cAMP levels, there was no significant difference when GHRGF concentration was in the range of 250-1000 μM. However, when the concentration reached 2000 μM, the intracellular cAMP concentration decreased significantly, by 30.18%. The arbutin group reduced the intracellular cAMP concentration by 35.85%. For extracellular cAMP concentration, treatment with 1000 μM and 2000 μM GHRGF reduced cAMP levels by 36.8% and 50.92%, respectively.

[0080] The effects of GHRGF on the cAMP / PKA / CREB signaling pathway:

[0081] The expression levels of mRNA of six genes, namely TYR, MITF, TRP-1, TRP-2, PKA and CREB, were detected by qRT-PCR, and the expression differences of related proteins were determined by Western blot.

[0082] like Figure 6 As shown, after GHRGF treatment of B16F10 cells, the expression levels of all six genes showed a decreasing trend, and the relationship was significantly dose-dependent.

[0083] like Figure 7 As shown, the expression of these six proteins was also significantly downregulated in B16F10 cells in a dose-dependent manner.

[0084] GHRGF may inhibit melanin synthesis by suppressing cAMP levels in cells, thereby inhibiting the activity of PKA and CREB, downregulating MITF expression, and ultimately suppressing the expression of melanin synthesis-related proteins TYR, TRP-1, and TRP-2. At the same time, the intracellular MDA content is significantly reduced, suggesting that GHRGF may also block melanin synthesis and transformation by alleviating intracellular oxidative stress.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a grass carp skin collagen peptide in the preparation of whitening products, characterized in that: The amino acid sequence of grass carp skin collagen peptide is Gly-His-Arg-Gly-Phe.

2. The application of a composition containing grass carp skin collagen peptides in the preparation of whitening products, characterized in that: The amino acid sequence of grass carp skin collagen peptide is Gly-His-Arg-Gly-Phe.

3. The application according to claim 2, characterized in that: The preparation method of the grass carp skin collagen peptide composition includes the following steps: S1: Pre-treatment of grass carp skin; S2: The fish skin pretreated in step S1 is swelled in acetic acid solution, pepsin is added for enzymatic hydrolysis, and after enzymatic hydrolysis, it is filtered and centrifuged. Salt is added to the supernatant while stirring, and after standing and centrifuging, the precipitate is collected, dissolved in acetic acid, dialyzed to obtain dialysate, and then freeze-dried under vacuum to obtain purified grass carp skin collagen. S3: Prepare a collagen solution by mixing the purified grass carp skin collagen from step S2 with a buffer solution; add collagenase for hydrolysis, boil the hydrolysate, cool to room temperature, and centrifuge to obtain grass carp skin collagen hydrolysate, which is the composition of grass carp skin collagen peptides.

4. The application according to claim 3, characterized in that: The purified collagen was prepared into a collagen solution with a mass concentration of 2.3-2.5% using buffer solution, and 7% collagenase (m / m) was added. The pH was adjusted to 7.4-7.3, and the solution was hydrolyzed at 41.7℃ for 4 hours.

5. The application according to claim 3, characterized in that: The pretreatment of grass carp skin in step S1 includes: A1: Thaw the frozen grass carp skin at room temperature, wash it, scrape off any remaining fish meat, scales and bones, cut it into small pieces of the same size, and put it in an oven to dry and remove moisture to obtain completely dried grass carp skin. A2: Cut the completely dried grass carp skin into small pieces, weigh the fish skin, soak it in n-butanol solution, change the n-butanol solution several times, rinse it clean with running water, and then treat it with NaOH solution to remove the impurities in the fish skin, thus obtaining the pretreated grass carp skin.

6. The application according to claim 5, characterized in that: The salt in the NaOH solution containing salt in step S2 is NaCl.

7. The application according to claim 5, characterized in that: After pretreatment in step S2, the grass carp skin was swollen in a 0.6-0.8 M acetic acid solution for 10-14 h at a material-to-liquid ratio of 1:55-65 g / mL.

8. The application according to claim 5, characterized in that: The amount of pepsin added is 1900-2100 U / g.

9. The application according to claim 5, characterized in that: In step S2, the swollen pepsin and acetic acid are stirred at 28-32℃ for 4-5 hours, filtered, and the filtrate is centrifuged at 3900-4100 r / min for 28-32 min, and the supernatant is retained.

10. The application according to claim 5, characterized in that: While slowly stirring, salt was added to the supernatant until the concentration reached 2.4-2.6 M. After standing for 20-24 h, the mixture was centrifuged at 3900-4100 r / min for 29-31 min, the liquid was discarded, and the precipitate was collected. The collected precipitate was redissolved in 0.4-0.6 M acetic acid, and dialyzed with running water for 46-50 h. The dialysate was collected and freeze-dried under vacuum until constant weight to obtain purified grass carp skin collagen.

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