Method for producing collagen from fish scales, product and application

Abstract

The invention belongs to the technical field of fish scale collagen preparation, and particularly relates to a method for producing collagen from fish scales. Comprising the following preparation steps: (1) pretreatment: (1) cleaning: soaking and cleaning fresh grass carp scales with pure water to remove attached fish body tissues, and naturally airing; (2) degreasing and impurity removal: adding the fish scales into a solution of alkali and alkali salt, fully stirring, and standing at room temperature for 0.5-10 hours; filtering out the fish scales by using filter cloth, adding pure water until the fish scales are immersed, uniformly stirring, standing at room temperature for 5 minutes, and repeating for 1-4 times; (3) decalcification: filtering out the fish scales by using filter cloth, adding the fish scales into an acid solution, uniformly stirring, and standing at room temperature for 0.5-10 hours; filtering out the fish scales by using filter cloth, adding pure water until the fish scales are immersed, uniformly stirring, standing at room temperature for 5 minutes, and repeating for 1-4 times to finish pretreatment; the fish scales are filtered out with filter cloth and then added into pure water, and an alkaline solution is added to adjust the pH value; and (4) cryopreservation: performing freeze-drying preservation on the obtained fish scales in a liquid nitrogen environment.

Description

Technical Field

[0001] The invention belongs to the technical field of fish scale collagen preparation, and particularly relates to a method for producing collagen by using fish scales. Background Art

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

[0003] my country is rich in aquatic resources. According to the National Fisheries and Fishery Administration, the total amount of aquatic products in 2021 reached 66.9029 million tons, a year-on-year increase of 2.16%. 80% are farmed products, a year-on-year increase of 3.26%. Among them, fish scales account for 1%-5% of body weight, which is equivalent to 670,000-3.34 million tons of raw materials to be fully utilized each year.

[0004] The extraction methods of fish scale protein mostly follow a process: washing, removing impurities and proteins, removing calcium salts, protein extraction, and purification. There are various methods used in each step, but most of them use water as a solvent due to cost advantages. At the same time, multiple extractions are performed to maximize protein extraction. The biggest problem caused by this is that the solid content of the solution after protein extraction is very low, only about 1%, and the low solid content requires a lot of cost to concentrate.

[0005] Commonly used concentration methods such as negative pressure concentration, centrifugation, and salting out have certain defects. Concentration requires corresponding equipment and takes a long time. Centrifugation also requires centrifugal equipment and pretreatment to separate the protein from the supernatant. Although the salting out method is simple, it requires the addition of a large amount of salt reagents and needs to be removed later.

[0006] In the example of the Chinese invention patent (publication number CN103468772A), the salt precipitation process requires the addition of inorganic salts with a concentration of 6-20% by volume of the extract, that is, 6-20% by mass of the extract, and then the inorganic salts need to be removed by membrane separation. The addition of salt reagents up to 6-20% by mass of the extract and the subsequent membrane separation and removal process will greatly increase the cost.

[0007] Divalent and trivalent metal salts have stronger ability to precipitate proteins, but some metal salts have problems such as toxicity and irreversible denaturation of proteins. Zinc salts do not have the above disadvantages. When combined with flocculants, proteins can be precipitated. Only 0.032% of the extract by mass of zinc salts and 0.16% of the flocculant by mass are needed to effectively precipitate proteins. The amount of reagents required for this method is only a few hundredths of that of the inorganic salt precipitation method, and therefore no subsequent desalination process is required. Summary of the invention

[0008] In order to solve the above 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 then dry naturally;

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

[0012] (3) decalcification: filter the fish scales with a filter cloth and then add them to an acid solution, stir well and place at room temperature for 0.5-10 hours; filter the fish scales with a filter cloth and then add pure water until they are submerged, stir well and place at room temperature for 5 minutes, repeat 1-4 times to complete the pretreatment; filter the fish scales with a filter cloth and then add them to pure water, and add an alkaline solution to adjust the pH value;

[0013] (4) Cryopreservation: freeze-drying and preserving the obtained fish scales in a liquid nitrogen environment;

[0014] Step 2: Extract collagen (1)

[0015] (1) crushing the frozen fish scales and mixing them with the solution, heating the solution for 0.5-4h for preliminary extraction, filtering and collecting the supernatant, and collecting the precipitate for further extraction;

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

[0017] (3) adding pure water to the precipitate, heating for 0.5-4 h for the third extraction, filtering and collecting the supernatant, and collecting the precipitate;

[0018] (4) combining the supernatants of the three extractions to obtain an extract;

[0019] Step 3: Extract collagen (2)

[0020] (2) The precipitate in step (3) of the second step and the plant extract solution were mixed and stirred for 48 hours, and filtered through a double-layer gauze to collect the collagen (2) solution; the collected solution was centrifuged at 9,000 r / min for 30 minutes and the supernatant was collected;

[0021] Step 4: Collagen Concentration

[0022] (1) mixing the supernatant and the extract to obtain a mixed solution;

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

[0024] (3) Add metal salt solution to the mixed solution at a ratio of 50 ml:1 mL, stir slowly for 30 seconds, and place at room temperature for 5-60 minutes. The collagen will spontaneously aggregate into clumps and separate from the supernatant. Filter 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 the 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 water, and tetramethylammonium, and the alkali salt is selected from one or more of sodium carbonate, sodium sulfate, sodium chloride, and sodium phosphate.

[0026] As a preferred embodiment of the present invention, in the solution of alkali and alkali salt, the concentration of alkali is 1-100 g / L, and the concentration of 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, and the concentration is 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 an aqueous solution of one or more selected from polyaluminum, polyiron, chitosan, chitin, polyacrylamide, and polyquaternary ammonium salt flocculants, with a concentration of 0.001-1 wt%.

[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 and zinc chloride, with a concentration of 0.1-0.2 mol / L.

[0032] A collagen is prepared by the method.

[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] The present invention provides an industrial method for producing collagen using fish scales as raw materials, using zinc salts to assist flocculants to precipitate proteins, and does not require concentration equipment, energy consumption or a large amount of reagents, so as to solve the problem of high concentration process cost in fish scale protein extraction methods. The collagen produced by the present invention has the effects of moisturizing, anti-wrinkle and firming, and can be used as a food additive or a cosmetic raw material. The concentration cost is low, no special equipment or energy consumption is required, and the reagent consumption is low, and the amount of reagent used is only a few hundredths of that of other precipitation methods. The time consumption is low, and the protein is separated from the supernatant and spontaneously aggregated into a mass in about 5-60 minutes after the flocculant and zinc salt are added. The concentration efficiency is high, and the solid content of the concentrated extract is increased from 1.15% to 3.82%, which is equivalent to evaporating 69.9% of the volume. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 Fish scales before processing;

[0038] Figure 2 The fish scales are defatted and cleaned;

[0039] Figure 3 It is the decalcified fish scale;

[0040] Figure 4 After adding flocculants and metal salt solutions to the extract, the proteins spontaneously aggregate into agglomerates and separate from the supernatant;

[0041] Figure 5 It is a collagen extract. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Embodiment 1:

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

[0045] 1) Cleaning: Soak fresh fish scales in pure water to remove attached fish tissues, and dry naturally;

[0046] 2) Degreasing and impurity removal: add into a solution of 40g / L sodium hydroxide and 100g / L sodium sulfate according to the solid-liquid ratio of 15g:100mL, stir well, and place at room temperature for 10h;

[0047] 3) Filter the fish scales with filter cloth and add pure water until they are submerged, stir evenly and leave at room temperature for 5 minutes, repeat 4 times;

[0048] 4) Filter the fish scales with filter cloth and add 15g:100mL into 20mL / L hydrochloric acid solution, stir well and place at room temperature for 10h;

[0049] 5) Filter the fish scales with filter cloth and add pure water until they are submerged, stir evenly and leave at room temperature for 5 minutes. Repeat twice to complete the pretreatment;

[0050] 6) filtering the fish scales with a filter cloth, and freeze-drying the obtained fish scales in a liquid nitrogen environment for storage;

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

[0052] The solution was heated at 60°C for 3 h for the first extraction, the supernatant was collected by filtration, and the precipitate was collected for further extraction;

[0053] 8) Add 15 g of the precipitate to 100 mL of pure water, heat at 70 °C for 3 h for the second extraction, filter and collect the supernatant, and collect the precipitate for further extraction;

[0054] 9) Add 15 g of the precipitate to 100 mL of pure water, heat at 80 °C for 3 h for the 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) The precipitate from step 9) and pure water were mixed and stirred for 48 hours, and then ultrasonically treated for 15 minutes at an ultrasonic frequency of 30 kHz and filtered with double-layer gauze to collect the collagen (2) solution; the collected solution was centrifuged at 9000 r / min for 30 minutes and the supernatant was collected;

[0057] 12) The extract obtained in step 10) and the supernatant obtained in step 11) were mixed, 0.1% polyquaternium-10 flocculant was added thereto at a ratio of 2 ml:1 mL, and the mixture was stirred slowly for 30 seconds;

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

[0059] Embodiment 2:

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

[0061] 1) Cleaning: Soak fresh fish scales in pure water to remove attached fish tissues, and dry naturally;

[0062] 2) Degreasing and impurity removal: add into a solution of 40g / L sodium hydroxide and 100g / L sodium sulfate according to the solid-liquid ratio of 15g:100mL, stir well, and place at room temperature for 10h;

[0063] 3) Filter the fish scales with filter cloth and add pure water until they are submerged, stir evenly and leave at room temperature for 5 minutes, repeat 4 times;

[0064] 4) Filter the fish scales with filter cloth and add 15g:100mL into 20mL / L hydrochloric acid solution, stir well and place at room temperature for 10h;

[0065] 5) Filter the fish scales with filter cloth and add pure water until they are submerged, stir evenly and leave at room temperature for 5 minutes. Repeat twice to complete the pretreatment;

[0066] 6) filtering the fish scales with a filter cloth, and freeze-drying the obtained fish scales in a liquid nitrogen environment for storage;

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

[0068] The solution was heated at 60°C for 3 h for the first extraction, the supernatant was collected by filtration, and the precipitate was collected for further extraction;

[0069] 8) Add 15 g of the precipitate to 100 mL of pure water, heat at 70 °C for 3 h for the second extraction, filter and collect the supernatant, and collect the precipitate for further extraction;

[0070] 9) Add 15 g of the precipitate to 100 mL of pure water, heat at 80 °C for 3 h for the third extraction, and 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 0.1 mol / L zinc sulfate solution to the extract at a ratio of 50 ml:1 mL, stir slowly for 30 seconds, and place at room temperature for 15 minutes. The collagen will spontaneously aggregate into clumps and separate from the supernatant. Filter 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, comprising the following steps:

[0076] 1) Cleaning: Soak fresh fish scales in pure water to remove attached fish tissues, and dry naturally;

[0077] 2) Degreasing and impurity removal: add into a solution of 40g / L sodium hydroxide and 100g / L sodium sulfate according to the solid-liquid ratio of 15g:100mL, stir well, and place at room temperature for 10h;

[0078] 3) Filter the fish scales with filter cloth and add pure water until they are submerged, stir evenly and leave at room temperature for 5 minutes, repeat 4 times;

[0079] 4) Filter the fish scales with filter cloth and add 15g:100mL into 20mL / L hydrochloric acid solution, stir well and place at room temperature for 10h;

[0080] 5) Filter the fish scales with filter cloth and add pure water until they are submerged, stir evenly and leave at room temperature for 5 minutes. Repeat twice to complete the pretreatment;

[0081] 6) filtering the fish scales with a filter cloth, and freeze-drying the obtained fish scales in a liquid nitrogen environment for storage;

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

[0083] The solution was heated at 60°C for 3 h for the first extraction, the supernatant was collected by filtration, and the precipitate was collected for further extraction;

[0084] 8) Add 15 g of the precipitate to 100 mL of pure water, heat at 70 °C for 3 h for the second extraction, filter and collect the supernatant, and collect the precipitate for further extraction;

[0085] 9) Add 15 g of the precipitate to 100 mL of pure water, heat at 80 °C for 3 h for the 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) The precipitate from step 9) and pure water were mixed and stirred for 48 hours, and then ultrasonically treated for 15 minutes at an ultrasonic frequency of 30 kHz and filtered with double-layer gauze to collect the collagen (2) solution; the collected solution was centrifuged at 9000 r / min for 30 minutes and the supernatant was collected;

[0088] 12) The extract obtained in step 10) and the supernatant obtained in step 11) were mixed, 0.1% polyquaternium-10 flocculant was added thereto at a ratio of 2 ml:1 mL, and the mixture was stirred slowly for 30 seconds;

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

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

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

[0092] Example 4

[0093] The present embodiment provides a method for producing collagen using fish scales, comprising the following steps:

[0094] 1) Soak fresh fish scales in pure water to remove attached fish tissues, and dry them naturally;

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

[0096] 3) Filter the fish scales with filter cloth and add pure water until they are submerged, stir evenly and leave at room temperature for 5 minutes, repeat 4 times;

[0097] 4) Filter the fish scales with filter cloth and add 15g:100mL into 2mol / L phosphoric acid and 0.5mol / L hydroxyethylidene-1,1-diphosphonic acid solution, stir well and place at room temperature for 10h;

[0098] 5) Filter the fish scales with filter cloth and add pure water until they are submerged, stir evenly and leave at room temperature for 5 minutes. Repeat twice to complete the pretreatment;

[0099] 6) Filter the fish scales with filter cloth and add 15g:100mL of pure water, add 20g / L calcium hydroxide and 20g / L zinc hydroxide solution to adjust the pH to 5.0;

[0100] 7) Heat the solution at 60°C for 3 h for the first extraction, collect the supernatant by filtration, and collect the precipitate for further extraction;

[0101] 8) Add 15 g of the precipitate to 100 mL of pure water, heat at 70 °C for 3 h for the 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) The precipitate from step 9) and pure water were mixed and stirred for 48 hours, and then ultrasonically treated for 15 minutes at an ultrasonic frequency of 30 kHz and filtered with double-layer gauze to collect the collagen (2) solution; the collected solution was centrifuged at 9000 r / min for 30 minutes and the supernatant was collected;

[0104] 12) The extract obtained in step 10) and the supernatant obtained in step 11) were mixed, 0.1% polyquaternium-10 flocculant was added thereto at a ratio of 2 ml:1 mL, and the mixture was stirred slowly for 30 seconds;

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

[0106] Performance Testing

[0107] Test 1: Repair efficacy (zebrafish tissue regeneration recovery rate test)

[0108] 1. Test Principle

[0109] The structure of zebrafish skin is similar to that of human skin, and the skin can heal itself after being damaged. The skin of the zebrafish tail fin is large, and surgical removal of part of it does not affect the normal activities of the fish, and it can regenerate, and it is easy to operate, observe and measure. By comparing the differences in the regeneration of the tail fin skin, we can evaluate whether the test substance has a repair effect.

[0110] Zebrafish larvae at the 30th day of development were selected as experimental organisms.

[0111] A blank control group and a sample group (0.1%) were set up, and 10 zebrafish were set up in each experimental group. After MS-222 anesthesia, they were laid flat on their sides in a sterile culture dish. Part of the tail fin tissue was removed with a surgical blade under a dissecting microscope without damaging the notochord, and placed in a 24-well plate. 2mL of the working solution corresponding to each concentration group was added to each well; the 24-well plate to be tested was placed in a 28.5±0.5℃ constant temperature incubator and incubated for 48h. After the incubation, MS-222 anesthesia was performed, and the fish were placed under a stereo microscope, lying on their sides and flattening the tail fin for photography. After the photography was completed, the new area of ​​the selected area of ​​the tail fin of each zebrafish was counted, and the mean and standard error of each group were calculated. SPSS software was used for variance analysis to compare the significance of the new area of ​​the tail fin among the groups, and p<0.05 was considered to be significantly different.

[0112] The promotion rate of the new area rate of the zebrafish tail fin (i.e., the repair promotion rate) was calculated using the following formula:

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

[0114] (1) Where:

[0115] T—the average value of the newly added area of ​​the zebrafish tail fin in the test group;

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

[0117] A two-tailed T test was performed on the new areas of the caudal fin of the zebrafish in the test group and the control group, and p<0.05 was considered different.

[0118] Table 1 Test results of test 1

[0119] Example Repair promotion rate% Example 1 23.37 Example 2 18.42 Example 3 21.35 Example 4 21.13

[0120] Test 2: Antioxidant test-DPPH free radical scavenging rate

[0121] The antioxidant capacity of the sample was evaluated by detecting the scavenging rate of the sample for DPPH free radicals. Anhydrous ethanol [analytical grade, GENERAL-REAGENT], 1,1-diphenyl-2-picrylhydrazyl (DPPH) [98%, source leaf]. The sample was prepared into a 10% aqueous solution, and 3 parallel samples were prepared. The reagents were added according to the reaction system in the table below, mixed, and reacted at room temperature in the dark for 30 minutes, and then the absorbance was measured at 519nm. The average value of the parallel samples was taken as the final result of the sample. When the scavenging rate is ≥50%, the sample is considered to have an antioxidant effect.

[0122] DPPH free radical scavenging rate = 1-(As-Ar) / (A0)×100%

[0123] Where: As is the absorbance value of sample solution-DPPH, Ar is the absorbance value of sample solution-anhydrous ethanol, and A0 is the absorbance value of DPPH-anhydrous ethanol.

[0124] Table 2 Test results of test 2

[0125]

[0126]

[0127] Test 3: Moisture Retention Test - Gravimetric Method

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

[0129] Place the prepared potassium carbonate saturated solution in a desiccator and adjust the required temperature and humidity (relative humidity is 48%, temperature is 26°C); place a glass plate with a 5cm×5cm 3M tape in it and balance it for at least 8 hours; weigh the mass M0 of the glass plate with a 5cm×5cm 3M tape with an analytical balance (accurate to 0.0001g); use a glass rod to weigh the sample at 8±0.1mg / cm 2 Apply evenly on a glass plate with adhesive tape, weigh the mass M1 with an analytical balance (accurate to 0.0001g), and then put it into a dryer with the required constant humidity; place it for 4h, 6h, and 8h, and weigh the glass plate coated with the sample at each time point. The test is considered to be over when the difference between the last weighing and the second-to-last weighing is less than 1% of the second-to-last mass. Calculate the moisture retention rate based on the last weighing mass, and record its mass as M2 (accurate to 0.0001g).

[0130] Each sample was tested three times in parallel, and the average value was reported with accuracy to one decimal place.

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

[0132] Where: M0 is the mass of the empty plate (g); M1 is the mass of the glass plate after adding the sample (g); M2 is the mass after being placed in the dryer for several 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: 0.5ml sample + 0.5ml hyaluronidase solution + 0.1ml 2.5mol / L CaCl2 solution

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

[0139] Negative group: 0.5ml purified water + 0.5ml hyaluronidase solution + 0.1ml 2.5mol / 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] The test tubes were placed at 37°C for 20 min; then the corresponding reagents were added.

[0142] Sample set: 0.5 ml hyaluronidase solution

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

[0144] Negative group: 0.5 ml hyaluronidase solution

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

[0146] The test tube was placed at 37°C for 40 min and then at room temperature for 10 min.

[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, place in a boiling water bath for 30 min, place in an ice bath for 10 min, and then place at room temperature for 10 min; add 1 ml of P-DAB color developer solution to each of the four groups of test tubes, mix well, shake thoroughly for 30 s, add anhydrous ethanol to 8 ml, place at room temperature for 30 min, and measure the absorbance at a wavelength of 530 nm using a spectrophotometer.

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

[0149] Wherein: 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; ODD is the absorbance of the negative control group.

[0150] Three parallel samples were set up for the sample group, and the average value of the parallel samples was taken to report the results. When the hyaluronidase inhibition rate of 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 0.1U / mL with Tris-HCl buffer, the substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide was dissolved and diluted to 1.0mg / mL, and EGCG was diluted to 80mg / mL. Prepare 3 parallel samples of the sample, add reagents according to the reaction system in the table below, mix well, react at room temperature for 30 minutes, and measure the absorbance at 410nm. The average value of the parallel samples was taken as the final result of the sample. When the inhibition rate of the sample group is ≥30% of the inhibition rate of the positive control group, the sample is considered to have an anti-wrinkle / firming effect.

[0155] Elastase activity inhibition rate formula = 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 Test results of 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 to 4 was added as a moisturizing ingredient to skin care products to obtain moisturizing lotions 1 to 4. The specific formulas were as follows in parts by mass:

[0161] 5 parts of isododecane

[0162] 6 parts of bifid yeast fermentation filtrate

[0163] 3 parts of Cetearyl Alcohol

[0164] 5 parts of polydimethylsiloxane

[0165] Ammonium acryloyldimethyltaurate / VP copolymer 0.76 parts

[0166] 0.5 parts of p-hydroxyacetophenone

[0167] 2 portions of collagen from Examples 1 to 4 respectively

[0168] Hyaluronic acid 5 parts

[0169] 1 part niacinamide

[0170] 0.5 parts of peony extract

[0171] DENDROBIUM CANDIDUM stem extract 0.5 parts

[0172] Panthenol 0.35 parts

[0173] (Daily use) 0.1 part of flavor

[0174] 0.05 parts of Ipomoea purpurea extract

[0175] 10 portions of ginseng root water

[0176] Water to 100 parts.

[0177] Store moisturizing lotions 1 to 4 at 5°C, 25°C and 40°C for 90 days respectively, and check the product properties.

[0178] Table 7 Test results of test 6

[0179] Example 5℃ 25℃ 40℃ Example 1 No change No change No change Example 2 Partial precipitation Slightly delaminated Partial precipitation Example 3 Slight precipitation No change Slight precipitation Example 4 Slight precipitation No change Slight precipitation

[0180] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 method comprises the following preparation steps: First step preprocessing (1) Cleaning: soak fresh grass carp scales in pure water to remove attached fish tissues, and then dry naturally; (2) Degreasing and removing impurities: Add fish scales to a solution of alkali and alkali salt, stir thoroughly, and place at room temperature for 0.5-10 hours; then filter out the fish scales with a filter cloth and add pure water until the scales are submerged, stir well, and place at room temperature for 5 minutes, repeating 1-4 times; (3) decalcification: filter the fish scales with a filter cloth and then add them to an acid solution, stir well and place at room temperature for 0.5-10 hours; filter the fish scales with a filter cloth and then add pure water until they are submerged, stir well and place at room temperature for 5 minutes, repeat 1-4 times to complete the pretreatment; filter the fish scales with a filter cloth and then add them to pure water, and add an alkaline solution to adjust the pH value; (4) Cryopreservation: freeze-drying and preserving the obtained fish scales in a liquid nitrogen environment; Step 2: Extract collagen (1) (1) crushing the frozen fish scales and mixing them with the solution, heating the solution for 0.5-4h for preliminary extraction, filtering and collecting the supernatant, and collecting the precipitate for further extraction; (2) Add pure water to the precipitate, heat for 0.5-4 h for a second extraction, filter and collect the supernatant, and collect the precipitate for further extraction; (3) adding pure water to the precipitate, heating for 0.5-4 h for the third extraction, filtering and collecting the supernatant, and collecting the precipitate; (4) combining the supernatants from the third extraction to obtain an extract; Step 3: Extract collagen (2) (1) mixing the precipitate in step (3) and the plant extract solution in step 2 and stirring for 48 hours, and filtering with double-layer gauze to collect the collagen (2) solution; The collected solution was centrifuged at 9000 r / min for 30 min and the supernatant was taken; Step 4: Collagen Concentration (1) mixing the supernatant and the extract to obtain a mixed solution; (2) Add flocculant to the mixture at a ratio of 2 ml:1 mL and stir slowly for 30 seconds; (3) Add metal salt solution to the mixed solution at a ratio of 50 ml:1 mL, stir slowly for 30 seconds, and place at room temperature for 5-60 minutes. The collagen will spontaneously aggregate into clumps and separate from the supernatant. Filter the supernatant and collect the precipitate to obtain the final product collagen.

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

4. The method for producing collagen using 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, and the concentration is 0.1-3 mol / L.

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

6. The method for producing collagen using 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. The method for producing collagen using fish scales according to claim 1, characterized in that: The flocculant in the fourth step (2) is selected from one or more aqueous solutions of polyaluminum, polyiron, chitosan, chitin, polyacrylamide, and polyquaternary ammonium salt flocculants, with a concentration of 0.001-1wt%.

8. The method for producing collagen using fish scales according to claim 1, characterized in that: The metal salt solution in the fourth step (3) is an aqueous solution of one or more of zinc sulfate and zinc chloride, with a concentration of 0.1-0.2 mol / L.

9. A collagen protein, characterized in that: The method is prepared by any one of claims 1 to 8.

10. The collagen according to claim 9, characterized in that Used in daily chemicals, agricultural chemicals and food fields.

Images