A method for producing collagen from fish scales, products, applications

Abstract

The application belongs to the technical field of fish scale collagen preparation, and particularly relates to a method for producing collagen from fish scales. The method comprises the following preparation steps: the first pretreatment (1) cleaning, fresh grass carp scales are cleaned by soaking in pure water to remove attached fish tissues, and then naturally dried; (2) degreasing and impurity removal, the scales are added into a solution of alkali and alkali salt, fully stirred, and then placed at room temperature for 0.5-10 h; then the scales are filtered out with filter cloth, and then pure water is added to immerse, stirred uniformly, and then placed at room temperature for 5 min, and the operation is repeated for 1-4 times; (3) decalcification, the scales are filtered out with filter cloth, and then added into an acid solution, stirred, and then placed at room temperature for 0.5-10 h; the scales are filtered out with filter cloth, and then pure water is added to immerse, stirred uniformly, and then placed at room temperature for 5 min, and the operation is repeated for 1-4 times to complete the pretreatment; the scales are filtered out with filter cloth, and then added into pure water, and an alkali solution is added to adjust the pH value; (4) freezing preservation, the obtained scales are frozen and dried in a liquid nitrogen environment for preservation.

Description

Technical Field

[0001] This invention belongs to the field of fish scale collagen preparation technology, specifically relating to a method for producing collagen using fish scales. Background Technology

[0002] Collagen is the most abundant structural protein in vertebrates and invertebrates, accounting for 30% of total protein content. Collagen and its derivatives are widely used in food, medicine, cosmetics, and other fields due to their nutritional value and functions. In recent years, due to safety concerns surrounding mammalian collagen, fish collagen and gelatin have received increasing attention.

[0003] my country is rich in aquatic resources. According to statistics from the State Fisheries and Fishery Administration, the total aquatic product output in 2021 reached 66.9029 million tons, an increase of 2.16% year-on-year. 80% of these were aquaculture products, with an increase of 3.26% year-on-year. Fish scales account for 1%-5% of the total body weight, which means that 670,000 to 3.34 million tons of raw materials are yet to be fully utilized each year.

[0004] Most fish scale protein extraction methods follow a single process: washing, removing impurities and proteins, removing calcium salts, protein extraction, and purification. Various methods are used in each step, but water is often used as the solvent due to cost advantages. Furthermore, multiple extractions are performed to maximize protein extraction. The biggest problem this causes is that the solid content of the extracted solution is very low, only about 1%, requiring significant investment in concentration.

[0005] Commonly used concentration methods, such as negative pressure concentration, centrifugation, and salting out, all have certain drawbacks. Concentration requires specific equipment and is time-consuming; centrifugation also requires centrifugation equipment and pretreatment to separate the protein from the supernatant; while salting out is simple, it requires the addition of a large amount of salt reagent, which needs to be removed subsequently.

[0006] In the example of the Chinese invention patent (publication number CN103468772A), the salting-out process requires the addition of inorganic salt at a concentration of 6-20% by volume of the extract, i.e., 6-20% by mass of inorganic salt in the extract. Furthermore, membrane separation is required to remove the inorganic salt afterward. The addition of such a high salt concentration (up to 6-20% by mass of the extract) and the subsequent membrane separation process significantly increase costs.

[0007] Divalent and trivalent metal salts have a stronger ability to precipitate proteins, but some metal salts are toxic and can cause irreversible protein denaturation. Zinc salts do not have these drawbacks. When used in conjunction with flocculants, only 0.032% by mass of zinc salt and 0.16% by mass of flocculant are needed to effectively precipitate proteins. This method requires only a fraction of the reagents needed for inorganic salt precipitation methods, and therefore eliminates the need for a subsequent desalination process. Summary of the Invention

[0008] To solve the above-mentioned technical problems, the present invention provides a method for producing collagen using fish scales, comprising the following preparation steps:

[0009] First step preprocessing

[0010] (1) Cleaning: Soak fresh grass carp scales in pure water to remove attached fish tissues, and let them air dry.

[0011] (2) Degreasing and removing impurities: Add fish scales to a solution of alkali and alkali salts, stir thoroughly, and let stand at room temperature for 0.5-10 hours; then filter out the fish scales with a filter cloth, add pure water until submerged, stir evenly, and let stand at room temperature for 5 minutes. Repeat 1-4 times.

[0012] (3) Decalcification: Filter the fish scales with a filter cloth and add them to the acid solution. Stir well and let stand at room temperature for 0.5-10 hours. Filter the fish scales with a filter cloth and add pure water until submerged. Stir well and let stand at room temperature for 5 minutes. Repeat 1-4 times to complete the pretreatment. Filter the fish scales with a filter cloth and add them to pure water. Add an alkaline solution to adjust the pH value.

[0013] (4) Freezing preservation: The obtained fish scales were freeze-dried and preserved in a liquid nitrogen environment;

[0014] Step 2: Extracting collagen (1)

[0015] (1) After the fish scales were frozen and stored, they were crushed and mixed with the solution. The solution was heated for 0.5-4 hours for preliminary extraction. The supernatant was collected by filtration and the precipitate was collected for further extraction.

[0016] (2) Add the precipitate to pure water, heat for 0.5-4 hours for a second extraction, filter and collect the supernatant, collect the precipitate and continue extraction;

[0017] (3) Add the precipitate to pure water, heat for 0.5-4 hours for a third extraction, filter and collect the supernatant, and collect the precipitate;

[0018] (4) Combine the supernatants from the three extractions to obtain the extract;

[0019] Step 3: Extract collagen (2)

[0020] (1) Mix the precipitate from step (3) in the second step with the plant extract solution and stir for 48 hours. Filter the mixture with double gauze and collect the collagen (2) solution. Centrifuge the collected solution at 9,000 r / min for 30 min and take the supernatant.

[0021] Step 4: Collagen Concentration

[0022] (1) Mix the supernatant and the extract to obtain a mixture;

[0023] (2) Add flocculant to the mixture at a ratio of 2ml:1mL and stir slowly for 30 seconds;

[0024] (3) Add metal salt solution to the mixture at a ratio of 50ml:1mL, stir slowly for 30 seconds, and let stand at room temperature for 5-60 minutes. The collagen will spontaneously aggregate into clusters and separate from the supernatant. Filter out the supernatant and collect the precipitate to obtain the final product collagen.

[0025] As a preferred embodiment of the present invention, in the solution of alkali and alkali salt in the first pretreatment step (2), the alkali is selected from one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, and tetramethylammonium, and the alkali salt is selected from one or more of sodium carbonate, sodium sulfate, sodium chloride, and sodium phosphate.

[0026] In a preferred embodiment of the present invention, the concentration of the alkali and alkali salt in the solution is 1-100 g / L and the concentration of the alkali salt is 1-150 g / L.

[0027] As a preferred embodiment of the present invention, the acid solution in the first pretreatment step (3) is selected from one or more aqueous solutions of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, citric acid, and tannic acid, with a concentration of 0.1-3 mol / L.

[0028] As a preferred embodiment of the present invention, the acid solution further comprises one or more of ethylenediaminetetraacetic acid (EDTA) disodium salt, hydroxyethylidene-1,1-diphosphonic acid (HEDP), and aminotrimethylenephosphonic acid (ATMP).

[0029] As a preferred embodiment of the present invention, the alkaline solution in the first pretreatment step (3) is selected from one or more aqueous solutions of sodium hydroxide, potassium hydroxide, calcium hydroxide, and ammonia water, with a concentration of 5-60 g / L and a pH range of 4-6.

[0030] As a preferred embodiment of the present invention, the flocculant in the fourth step (2) is selected from one or more aqueous solutions of polyaluminum, polyferric, chitosan, chitin, polyacrylamide, and polyquaternary ammonium salt flocculants, with a concentration of 0.001-1wt%.

[0031] As a preferred embodiment of the present invention, the metal salt solution in the fourth step (3) is an aqueous solution of one or more of zinc sulfate or zinc chloride, with a concentration of 0.1-0.2 mol / L.

[0032] A type of collagen, prepared by the method described above.

[0033] As a preferred embodiment of the present invention, the collagen is applied in the fields of daily chemicals, agricultural chemicals, and food.

[0034] By adopting the above technical solution, the present invention has the following beneficial effects:

[0035] This invention provides an industrial method for producing collagen from fish scales. It employs a zinc salt-assisted flocculant to precipitate proteins, eliminating the need for concentration equipment, energy consumption, or large amounts of reagents, thus solving the problem of high concentration costs in traditional fish scale protein extraction methods. Furthermore, the collagen produced by this invention has moisturizing, hydrating, anti-wrinkle, and firming effects, and can be used as a food additive or cosmetic ingredient. The concentration cost is low, requiring no special equipment or energy, and reagent consumption is minimal, only a fraction of that used in other precipitation methods. The time consumption is also low; after adding the flocculant and zinc salt, the protein spontaneously aggregates into clumps within approximately 5-60 minutes, separating from the supernatant. The concentration efficiency is high; the solid content of the concentrated extract increases from 1.15% to 3.82%, equivalent to an evaporation of 69.9% of the volume. Attached Figure Description

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 Fish scales before processing;

[0038] Figure 2 Fish scales after degreasing and impurity removal;

[0039] Figure 3 These are decalcified fish scales;

[0040] Figure 4 After adding flocculant and metal salt solution to the extract, the protein spontaneously aggregates into clusters and is separated from the supernatant.

[0041] Figure 5 It is a collagen extract. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. 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.

[0043] Example 1:

[0044] This embodiment provides a method for producing collagen using fish scales, including the following steps:

[0045] 1) Cleaning: Soak fresh fish scales in pure water to remove attached fish tissue, then air dry.

[0046] 2) Degreasing and impurity removal: Add the solid to liquid solution at a ratio of 15g:100mL to a solution of 40g / L sodium hydroxide and 100g / L sodium sulfate, stir thoroughly, and let stand at room temperature for 10 hours;

[0047] 3) Filter out the fish scales with a filter cloth, then add pure water until submerged, stir well and let stand at room temperature for 5 minutes. Repeat 4 times.

[0048] 4) Filter the fish scales through a filter cloth and add them to a 20 mL / L hydrochloric acid solution at a ratio of 15 g: 100 mL. Stir well and let stand at room temperature for 10 hours.

[0049] 5) Filter out the fish scales with a filter cloth, then add pure water until submerged, stir well and let stand at room temperature for 5 minutes. Repeat twice to complete the pretreatment.

[0050] 6) Filter the fish scales through a filter cloth, and freeze-dry the obtained fish scales in a liquid nitrogen environment for preservation;

[0051] 7) After crushing the frozen fish scales, mix them with the solution, add pure water at a ratio of 15g:100mL, and adjust the pH to 5.0 by adding 40g / L sodium hydroxide solution;

[0052] The solution was heated at 60℃ for 3 hours for the first extraction. The supernatant was collected by filtration, and the precipitate was collected for further extraction.

[0053] 8) Add the precipitate to pure water at a ratio of 15g:100mL, heat at 70℃ for 3h for a second extraction, filter and collect the supernatant, collect the precipitate and continue extraction;

[0054] 9) Add the precipitate to pure water at a ratio of 15g:100mL, heat at 80℃ for 3h for a third extraction, filter and collect the supernatant, and store the precipitate;

[0055] 10) Combine the supernatants from the three extractions to obtain the extract.

[0056] 11) Mix the precipitate from step 9) with pure water and stir for 48 hours, then sonicate at a frequency of 30 kHz for 15 minutes and filter with double gauze to collect the collagen (2) solution; centrifuge the collected solution at 9000 r / min for 30 min and take the supernatant.

[0057] 12) Mix the extract obtained in step 10) and the supernatant obtained in step 11), add 0.1% polyquaternium-10 flocculant at a ratio of 2 ml: 1 mL, and stir slowly for 30 seconds;

[0058] 13) Add 0.1mol / L zinc sulfate solution to the extract at a ratio of 50ml:1mL, stir slowly for 30 seconds, and let stand at room temperature for 15 minutes. The collagen will spontaneously aggregate into clusters and separate from the supernatant. Filter out the supernatant and collect the precipitate to obtain the final product, collagen.

[0059] Example 2:

[0060] This embodiment provides a method for producing collagen using fish scales, including the following steps:

[0061] 1) Cleaning: Soak fresh fish scales in pure water to remove attached fish tissue, then air dry.

[0062] 2) Degreasing and impurity removal: Add the solid to liquid solution at a ratio of 15g:100mL to a solution of 40g / L sodium hydroxide and 100g / L sodium sulfate, stir thoroughly, and let stand at room temperature for 10 hours;

[0063] 3) Filter out the fish scales with a filter cloth, then add pure water until submerged, stir well and let stand at room temperature for 5 minutes. Repeat 4 times.

[0064] 4) Filter the fish scales through a filter cloth and add them to a 20 mL / L hydrochloric acid solution at a ratio of 15 g: 100 mL. Stir well and let stand at room temperature for 10 hours.

[0065] 5) Filter out the fish scales with a filter cloth, then add pure water until submerged, stir well and let stand at room temperature for 5 minutes. Repeat twice to complete the pretreatment.

[0066] 6) Filter the fish scales through a filter cloth, and freeze-dry the obtained fish scales in a liquid nitrogen environment for preservation;

[0067] 7) After crushing the frozen fish scales, mix them with the solution, add pure water at a ratio of 15g:100mL, and adjust the pH to 5.0 by adding 40g / L sodium hydroxide solution;

[0068] The solution was heated at 60℃ for 3 hours for the first extraction. The supernatant was collected by filtration, and the precipitate was collected for further extraction.

[0069] 8) Add the precipitate to pure water at a ratio of 15g:100mL, heat at 70℃ for 3h for a second extraction, filter and collect the supernatant, collect the precipitate and continue extraction;

[0070] 9) Add the precipitate to pure water at a ratio of 15g:100mL, heat at 80℃ for 3h for a third extraction, filter and collect the supernatant;

[0071] 10) Combine the supernatants from the three extractions to obtain the extract.

[0072] 11) Add 0.1% polyquaternium-10 flocculant to the extract obtained in step 10) at a ratio of 2 ml: 1 mL, and stir slowly for 30 seconds;

[0073] 12) Add 0.1mol / L zinc sulfate solution to the extract at a ratio of 50ml:1mL, stir slowly for 30 seconds, and let stand at room temperature for 15 minutes. The collagen will spontaneously aggregate into clusters and separate from the supernatant. Filter out the supernatant and collect the precipitate to obtain the final product, collagen.

[0074] Example 3

[0075] This embodiment provides a method for producing collagen using fish scales, including the following steps:

[0076] 1) Cleaning: Soak fresh fish scales in pure water to remove attached fish tissue, then air dry.

[0077] 2) Degreasing and impurity removal: Add the solid to liquid solution at a ratio of 15g:100mL to a solution of 40g / L sodium hydroxide and 100g / L sodium sulfate, stir thoroughly, and let stand at room temperature for 10 hours;

[0078] 3) Filter out the fish scales with a filter cloth, then add pure water until submerged, stir well and let stand at room temperature for 5 minutes. Repeat 4 times.

[0079] 4) Filter the fish scales through a filter cloth and add them to a 20 mL / L hydrochloric acid solution at a ratio of 15 g: 100 mL. Stir well and let stand at room temperature for 10 hours.

[0080] 5) Filter out the fish scales with a filter cloth, then add pure water until submerged, stir well and let stand at room temperature for 5 minutes. Repeat twice to complete the pretreatment.

[0081] 6) Filter the fish scales through a filter cloth, and freeze-dry the obtained fish scales in a liquid nitrogen environment for preservation;

[0082] 7) After crushing the frozen fish scales, mix them with the solution, add pure water at a ratio of 15g:100mL, and adjust the pH to 5.0 by adding 40g / L sodium hydroxide solution;

[0083] The solution was heated at 60℃ for 3 hours for the first extraction. The supernatant was collected by filtration, and the precipitate was collected for further extraction.

[0084] 8) Add the precipitate to pure water at a ratio of 15g:100mL, heat at 70℃ for 3h for a second extraction, filter and collect the supernatant, collect the precipitate and continue extraction;

[0085] 9) Add the precipitate to pure water at a ratio of 15g:100mL, heat at 80℃ for 3h for a third extraction, filter and collect the supernatant, and store the precipitate;

[0086] 10) Combine the supernatants from the three extractions to obtain the extract.

[0087] 11) Mix the precipitate from step 9) with pure water and stir for 48 hours, then sonicate at a frequency of 30 kHz for 15 minutes and filter with double gauze to collect the collagen (2) solution; centrifuge the collected solution at 9000 r / min for 30 min and take the supernatant.

[0088] 12) Mix the extract obtained in step 10) and the supernatant obtained in step 11), add 0.1% polyquaternium-10 flocculant at a ratio of 2 ml: 1 mL, and stir slowly for 30 seconds;

[0089] 13) Add 0.1mol / L zinc sulfate solution to the extract at a ratio of 50ml:1mL, stir slowly for 30 seconds, and let stand at room temperature for 15 minutes. The collagen will spontaneously aggregate into clusters and separate from the supernatant. Filter out the supernatant and collect the precipitate to obtain the first-stage collagen extract.

[0090] 14) Add 0.1% cationic flocculant to the supernatant at a ratio of 2ml:1mL, and stir slowly for 30 seconds;

[0091] 15) Add 0.1mol / L zinc sulfate solution to the supernatant at a ratio of 50ml:1mL, stir slowly for 30 seconds, and let stand at room temperature for 15 minutes. The collagen will spontaneously aggregate into clumps and separate from the supernatant. Filter out the supernatant and collect the precipitate. Combine the precipitate with the collagen extracted in the first extraction to obtain the final product collagen peptone.

[0092] Example 4

[0093] This embodiment describes a method for producing collagen using fish scales, comprising the following steps:

[0094] 1) Soak fresh fish scales in pure water to wash away any attached fish tissue, then let them air dry naturally;

[0095] 2) Add the solution to 35 mL of ammonia water and 100 g / L sodium sulfate at a solid-liquid ratio of 15 g: 100 mL, stir thoroughly, and let stand at room temperature for 10 h;

[0096] 3) Filter out the fish scales with a filter cloth, then add pure water until submerged, stir well and let stand at room temperature for 5 minutes. Repeat 4 times.

[0097] 4) Filter the fish scales through a filter cloth and add them to a solution of 2 mol / L phosphoric acid and 0.5 mol / L hydroxyethylidene-1,1-diphosphonic acid at a ratio of 15 g to 100 mL. Stir well and let stand at room temperature for 10 h.

[0098] 5) Filter out the fish scales with a filter cloth, then add pure water until submerged, stir well and let stand at room temperature for 5 minutes. Repeat twice to complete the pretreatment.

[0099] 6) Filter the fish scales through a filter cloth and add them to pure water at a ratio of 15g:100mL. Adjust the pH to 5.0 by adding 20g / L calcium hydroxide and 20g / L zinc hydroxide solution.

[0100] 7) Heat the solution at 60℃ for 3 hours for the first extraction, filter and collect the supernatant, collect the precipitate and continue extraction;

[0101] 8) Add the precipitate to pure water at a ratio of 15g:100mL, heat at 70℃ for 3h for a second extraction, filter and collect the supernatant, and store the precipitate;

[0102] 10) Combine the supernatants from the three extractions to obtain the extract.

[0103] 11) Mix the precipitate from step 9) with pure water and stir for 48 hours, then sonicate at a frequency of 30 kHz for 15 minutes and filter with double gauze to collect the collagen (2) solution; centrifuge the collected solution at 9000 r / min for 30 min and take the supernatant.

[0104] 12) Mix the extract obtained in step 10) and the supernatant obtained in step 11), add 0.1% polyquaternium-10 flocculant at a ratio of 2 ml: 1 mL, and stir slowly for 30 seconds;

[0105] 13) Add 0.1mol / L zinc sulfate solution to the extract at a ratio of 50ml:1mL, stir slowly for 30 seconds, and let stand at room temperature for 15 minutes. The collagen will spontaneously aggregate into clusters and separate from the supernatant. Filter out the supernatant and collect the precipitate to obtain collagen.

[0106] Performance testing

[0107] Test 1: Repair Efficacy (Zebrafish Tissue Regeneration and Recovery Rate Test)

[0108] 1. Testing Principle

[0109] Zebrafish skin has a structure similar to human skin, and it can heal itself after injury. The skin area of ​​a zebrafish's caudal fin is relatively large; surgical removal of part of it does not affect the fish's normal activity, it can regenerate, and it is easy to manipulate and observe. By comparing the differences in caudal fin skin regeneration, the reparative efficacy of the test substance can be evaluated.

[0110] Zebrafish juveniles that had reached the 30-day mark were selected as the experimental organisms.

[0111] A blank control group and a sample group (0.1%) were set up, with 10 zebrafish in each experimental group. After anesthetizing with MS-222, the zebrafish were laid flat on their sides in sterile culture dishes. Under a dissecting microscope, part of the caudal fin tissue was removed with a scalpel without damaging the notochord, and the zebrafish were placed in 24-well plates. 2 mL of the working solution corresponding to each concentration group was added to each well. The 24-well plates were placed in a constant temperature incubator at 28.5±0.5℃ for 48 h. After incubation, the zebrafish were anesthetized with MS-222 and then photographed under a stereomicroscope with their caudal fins laid flat on their sides. After photographing, the newly added area of ​​the selected region of the caudal fin of each zebrafish was counted, and the mean and standard error of each group were calculated. SPSS software was used for analysis of variance to compare the significance of the differences in the newly added caudal fin area among the groups. p<0.05 was considered statistically significant.

[0112] The formula for calculating the rate of increase in caudal fin area (i.e., the repair promotion rate) in zebrafish is as follows:

[0113] Repair promotion rate (%) = (TC) / C × 100%

[0114] (1) In the formula:

[0115] T—The average increase in caudal fin area of ​​the zebrafish in the test group;

[0116] C—The average newly added area of ​​the caudal fin in the control group zebrafish;

[0117] Two-tailed t-tests were performed to compare the increased caudal fin area of ​​zebrafish in the test group and the control group, with p < 0.05 indicating a significant difference.

[0118] Table 1 Test Results of Test 1

[0119]

[0120]

[0121] Test 2: Antioxidant Test - DPPH Free Radical Scavenging Rate

[0122] The antioxidant capacity of samples was assessed by detecting their DPPH free radical scavenging rate. Anhydrous ethanol [analytical grade, general-reagent] and 1,1-diphenyl-2-picrylhydrazine (DPPH) [98%, source material] were used. The samples were prepared as a 10% aqueous solution, and three parallel samples were prepared. Reagents were added according to the reaction system shown in the table below, mixed well, and reacted at room temperature in the dark for 30 min. The absorbance was measured at 519 nm. The average value of the parallel samples was taken as the final result. A scavenging rate ≥50% was considered to indicate that the sample had an antioxidant effect.

[0123] DPPH radical scavenging rate = 1 - (As - Ar) / (A0) × 100%

[0124] In the formula: As is the absorbance of sample solution-DPPH, Ar is the absorbance of sample solution-anhydrous ethanol, and A0 is the absorbance of DPPH-anhydrous ethanol.

[0125] Table 2 Test Results of Test 2

[0126] Example Clearance rate (%) Example 1 59.87 Example 2 51.32 Example 3 57.35 Example 4 55.12

[0127] Test 3: Moisturizing Rate Test - Weight Method

[0128] The sample was evenly coated onto a biomaterial tape simulating the stratum corneum and skin. After being placed under constant temperature and humidity conditions for a period of time, the mass difference before and after was weighed to calculate the sample's moisturizing ability (moisturizing rate).

[0129] Place the prepared saturated potassium carbonate solution in a desiccator and adjust the temperature and humidity to the required levels (48% relative humidity, 26°C). Place a glass plate with 5cm x 5cm 3M tape on it and allow it to equilibrate for at least 8 hours. Weigh the glass plate with the 5cm x 5cm 3M tape (M0) using an analytical balance (accurate to 0.0001g). Use a glass rod to measure the sample at a concentration of 8 ± 0.1 mg / cm³. 2 The sample is evenly coated onto a glass plate covered with adhesive tape. The mass M1 (accurate to 0.0001g) is weighed using an analytical balance and then placed in a desiccator with constant humidity. After 4h, 6h, and 8h, the weight of the glass plate coated with the sample is weighed at each time point until the difference between the mass of the last weighing and the second-to-last weighing is less than 1% of the mass of the second-to-last weighing. The test is considered to be over when the mass of the last weighing is used to calculate the moisture retention rate, and the mass is recorded as M2 (accurate to 0.0001g).

[0130] Each sample was tested in parallel three times, and the average value was taken to report the result, accurate to one decimal place.

[0131] Moisturizing rate % = (M2 - M0) / (M1 - M0) × 100%

[0132] In the formula: M0 is the mass of the empty plate (g); M1 is the mass of the glass plate after the sample is added (g); M2 is the mass after the sample has been placed in the desiccator for a certain number of hours (g).

[0133] Table 3 Test Results of Test 3

[0134] Example Moisturizing rate (%) Example 1 37.8 Example 2 32.4 Example 3 35.6 Example 4 35.1 Positive control (5% glycerol aqueous solution) 28.7

[0135] Test 4: Soothing Efficacy Test - Hyaluronidase Inhibition Test

[0136] Take clean test tubes and label them as sample group, sample control group, negative group, and negative control;

[0137] Sample group: 0.5 ml sample + 0.5 ml hyaluronidase solution + 0.1 ml 2.5 mol / L CaCl2 solution

[0138] Sample control group: 0.5 ml sample + 0.5 ml sodium acetate buffer + 0.1 ml 2.5 mol / L CaCl2 solution

[0139] Negative control group: 0.5 ml purified water + 0.5 ml hyaluronidase solution + 0.1 ml 2.5 mol / L CaCl2 solution

[0140] Negative control: 0.5 ml purified water + 0.5 ml sodium acetate buffer + 0.1 ml 2.5 mol / L CaCl2 solution

[0141] Place the test tube at 37°C for 20 minutes; then add the appropriate reagents.

[0142] Sample group: 0.5 ml of hyaluronidase solution

[0143] Sample control group: 0.5 ml sodium acetate buffer

[0144] Negative group: 0.5ml hyaluronidase solution

[0145] Negative control: 0.5 ml sodium acetate buffer.

[0146] Place the test tube at 37°C for 40 minutes, then at room temperature for 10 minutes.

[0147] Add 0.5 ml of purified water, 0.1 ml of 5 mol / L NaOH solution, and 0.5 ml of acetylacetone solution to each test tube, mix well, incubate in a boiling water bath for 30 min, in an ice bath for 10 min, and then place at room temperature for 10 min. Add 1 ml of P-DAB colorimetric reagent solution to each of the four test tubes, mix well, shake thoroughly for 30 s, add anhydrous ethanol to 8 ml, and after placing at room temperature for 30 min, measure the absorbance at a wavelength of 530 nm using a spectrophotometer.

[0148] Hyaluronidase inhibition rate (%) = [(ODC-ODD)-(ODA-ODB)] / (ODC-ODD)×100%

[0149] In the formula: ODA is the absorbance of the sample group solution; ODB is the absorbance of the sample control group; ODC is the absorbance of the negative group solution; and ODD is the absorbance of the negative control group.

[0150] Three parallel samples were set up for the sample group. The average value of the parallel samples was taken and the results were reported. When the inhibition rate of hyaluronidase in the sample group was ≥50%, the sample was considered to have a soothing effect.

[0151] Table 4. Test Results of Test 4

[0152] Example Inhibition rate (%) Example 1 53.56 Example 2 42.41 Example 3 49.32 Example 4 49.13

[0153] Test 5: Anti-wrinkle / firming efficacy test - elastase inhibition test

[0154] Elastase was diluted to 0.1 U / mL using Tris-HCl buffer. The substrate N-succinyl-Ala-Ala-Ala-p-nitroaniline was dissolved and diluted to 1.0 mg / mL, and EGCG was diluted to 80 mg / mL. Three parallel samples were prepared. Reagents were added according to the reaction system in the table below, mixed well, and reacted at room temperature for 30 min. The absorbance was measured at 410 nm. The average value of the parallel samples was taken as the final result. When the inhibition rate of the sample group was ≥ 30% of the inhibition rate of the positive control group, the sample was considered to have an anti-wrinkle / firming effect.

[0155] The formula for elastase activity inhibition rate is: 1 - (OD sample group / positive control group - OD blank group) / (OD model control group - OD blank group) × 100%

[0156] Table 5 Sample preparation for Test 5

[0157]

[0158] Table 6 shows the test results for Test 5.

[0159] Example Inhibition rate (%) Example 1 50.35 Example 2 38.21 Example 3 43.16 Example 4 42.45 Positive control 29.27

[0160] Test 6: The collagen prepared in Examples 1-4 was used as a moisturizing ingredient and added to skincare products to obtain moisturizing lotions 1-4. The specific formulas are as follows, according to parts by weight:

[0161] 5 parts of isododecane

[0162] Six portions of Bifida ferment filtrate

[0163] 3 parts cetearyl alcohol

[0164] 5 parts of polydimethylsiloxane

[0165] 0.76 parts of ammonium acryloyldimethyltaurate / VP copolymer

[0166] 0.5 parts of p-hydroxyacetophenone

[0167] Two portions of collagen from Examples 1-4 respectively.

[0168] 5 parts hyaluronic acid

[0169] 1 part nicotinamide

[0170] 0.5 parts of peony extract

[0171] 0.5 parts of Dendrobium candidum stem extract

[0172] Panthenol 0.35 parts

[0173] (Daily Use) Fragrance 0.1 parts

[0174] 0.05 parts of Ipomoea purulenta extract

[0175] 10 portions of ginseng root water

[0176] Water to 100 servings.

[0177] Moisturizing lotions 1-4 were stored at 5℃, 25℃, and 40℃ for 90 days respectively, and the product properties were observed.

[0178] Table 7 shows the test results for Test 6.

[0179] Example 5℃ 25℃ 40℃ Example 1 No change No change No change Example 2 Partial sedimentation Slight stratification Partial sedimentation Example 3 Slight sedimentation No change Slight sedimentation Example 4 Slight sedimentation No change Slight sedimentation

[0180] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing collagen using fish scales, characterized in that, The preparation steps include the following: First step preprocessing (1) Cleaning: Soak fresh grass carp scales in pure water to remove attached fish tissues, and air dry. (2) Degreasing and removing impurities: Add fish scales to a solution of alkali and alkali salts, stir thoroughly, and let stand at room temperature for 0.5-10 hours; then filter the fish scales with a filter cloth, add pure water until submerged, stir evenly, and let stand at room temperature for 5 minutes. Repeat 1-4 times. (3) Decalcification: Filter the fish scales with a filter cloth and add them to the acid solution. Stir well and let stand at room temperature for 0.5-10 hours. Filter the fish scales with a filter cloth and add pure water until submerged. Stir well and let stand at room temperature for 5 minutes. Repeat 1-4 times to complete the pretreatment. Filter the fish scales with a filter cloth. (4) Freezing preservation: The obtained fish scales were freeze-dried and preserved in a liquid nitrogen environment; Step 2: Extracting collagen (1) (1) After the frozen fish scales are crushed, they are mixed with the solution and pure water is added at a ratio of 15g:100mL. The pH is adjusted to 5.0 by adding 40g / L sodium hydroxide solution. Heat the solution for 0.5-4 hours for preliminary extraction, filter and collect the supernatant, collect the precipitate and continue extraction; (2) Add the precipitate to pure water, heat for 0.5-4 hours for a second extraction, filter and collect the supernatant, collect the precipitate and continue extraction; (3) Add the precipitate to pure water, heat for 0.5-4 hours for a third extraction, filter and collect the supernatant, and collect the precipitate; (4) Combine the supernatants from the first three extractions to obtain the extract; Step 3: Extract collagen (2) (1) Mix the precipitate from step (3) in the second step with pure water and stir for 48 hours, then filter with double gauze to collect the collagen (2) solution; Centrifuge the collected solution at 9000 r / min for 30 min and take the supernatant; Step 4: Collagen Concentration (1) Mix the supernatant and the extract to obtain a mixture; (2) Add flocculant to the mixture at a ratio of 2ml:1mL and stir slowly for 30 seconds; (3) Add metal salt solution to the mixture at a ratio of 50ml:1mL, stir slowly for 30 seconds, and let stand at room temperature for 5-60 minutes. The collagen will spontaneously aggregate into clusters and separate from the supernatant. Filter out the supernatant and collect the precipitate to obtain the final product collagen. The flocculant in the fourth step (2) is selected from one or more aqueous solutions of polyaluminum, polyferric, chitosan, chitin, polyacrylamide, and polyquaternary ammonium salt flocculants, with a concentration of 0.001-1 wt%. The metal salt solution in the fourth step (3) is an aqueous solution of one or more of zinc sulfate or zinc chloride, with a concentration of 0.1-0.2 mol / L.

2. The method for producing collagen from fish scales according to claim 1, characterized in that, In the solution of alkali and alkali salt in the first step of pretreatment step (2), the alkali is selected from one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, and tetramethylammonium, and the alkali salt is selected from one or more of sodium carbonate, sodium sulfate, sodium chloride, and sodium phosphate.

3. The method for producing collagen from fish scales according to claim 1, characterized in that, In the solution of the alkali and alkali salt: the concentration of the alkali is 1-100 g / L, and the concentration of the alkali salt is 1-150 g / L.

4. The method for producing collagen from fish scales according to claim 1, characterized in that, The acid solution in the first pretreatment step (3) is selected from one or more aqueous solutions of hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid, citric acid and tannic acid, with a concentration of 0.1-3 mol / L.

5. The method for producing collagen from fish scales according to claim 4, characterized in that, The acid solution also contains one or more of the following: disodium ethylenediaminetetraacetic acid (EDTA), hydroxyethylidene-1,1-diphosphonic acid (HEDP), and aminotrimethylenephosphonic acid (ATMP).

6. The method for producing collagen from fish scales according to claim 1, characterized in that, The alkaline solution in the first pretreatment step (3) is selected from one or more aqueous solutions of sodium hydroxide, potassium hydroxide, calcium hydroxide, and ammonia water, with a concentration of 5-60 g / L and a pH range of 4-6.

7. A type of collagen, characterized in that, It is prepared by the method described in any one of claims 1 to 6.

8. The collagen according to claim 7 can be applied in the fields of daily chemicals, agricultural chemicals, and food.

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