Preparation method and application of squid collagen peptide with effects of maintaining beauty and keeping young

Abstract

The invention discloses a preparation method and application of squid collagen peptide with beauty maintaining and young keeping effects, and belongs to the technical field of marine food processing. The collagen is obtained through enzymolysis hydrolysis, the structure of the collagen is kept, damage to amino acid is avoided, treatment and use of acid, alkali and organic solvents are reduced in the preparation process, and equipment loss and environmental pollution are avoided. The second enzymolysis is performed by adopting the synergistic effect of the compound protease, the low molecular weight, degreasing and bitterness removal of the peptide powder can be realized, and the obtained peptide powder is low in molecular weight, good in flavor, free of peculiar smell and suitable for being eaten, digested and absorbed by human bodies. Experiments prove that the squid collagen peptide prepared by the preparation method disclosed by the invention has relatively strong oxidation resistance and has remarkable effects in the aspects of whitening, wrinkle resistance, oxidation resistance and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine food processing. Specifically, it particularly relates to a preparation method and application of squid collagen peptide with beauty and skin care effects. Background Art

[0002] Collagen is a biological macromolecular protein and an important component of tissues such as skin and bones. As people age, the collagen in the body will gradually be lost, leading to problems such as loose skin and increased wrinkles. Collagen peptide is made by degrading macromolecular collagen with protease, which has high digestibility, certain nutritional value, and beauty effects such as whitening and anti-wrinkle, and has received extensive attention from consumers in recent years. Currently, the conventional method is to prepare collagen peptide through processes such as high-temperature cooking and hydrolysis. However, the existing technology often requires repeated treatments with acids and alkalis for swelling, etc., the process is cumbersome, the purity is poor, the content of low-molecular-weight peptide components is not high, the functionality of the obtained peptide is poor, and the market application prospect is general.

[0003] Squid is an important marine resource with a large annual output and high nutritional value. Squid skin, as a waste in the processing process, is often used for low-value products such as feed and waste. Squid skin is a high-protein and low-fat raw material, containing rich collagen, vitamins, and minerals. There is no risk of animal-source diseases such as those from pigs, cows, and chickens. A high-value-added squid collagen peptide can be developed to improve its utilization rate and reduce environmental pollution and resource waste.

[0004] CN201210076538.1 discloses a preparation method of low-molecular-weight collagen peptide from squid skin. After the squid skin is mashed, it is treated by soaking in alkali for 24 - 30h, stirring with n-butanol for 16 - 24h, and treating with dilute acid for 6 - 8h. The crude extract is obtained and then prepared by enzymatic hydrolysis, filtration, decolorization, de-bittering, concentration, and drying; CN202311857505.5 discloses a preparation method of fish-derived collagen peptide. The fish body tissue is used to extract collagen by enzymatic hydrolysis at low temperature for 12 - 48h and then purified, and then collagen peptide is extracted by low-temperature acid-alcohol under long-time high-pressure homogenization. The preparation processes of both of them involve long-time acid / alkali and organic solvent treatments, with high requirements for process equipment, long production cycles, and are not suitable for large-scale production. CN202410699900.3 discloses a preparation method and application of fresh collagen peptide with anti-aging and antioxidant effects. It pre-treats the raw material by swelling with acid and alkali, then performs the first enzymatic hydrolysis with acidic protease, and then performs the second enzymatic hydrolysis with compound protease to obtain fresh collagen. The raw material is defatted by treatment with a large amount of alkali solution, and the product properties are not conducive to storage and transportation. Summary of the Invention

[0005] The object of the present invention is to provide a preparation method of squid collagen peptide with beauty and skin care effects, and another object is to provide the specific application of the protein peptide to make up for the deficiencies of the prior art.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] A preparation method of squid collagen peptide with beauty and skin care effects, comprising the following steps:

[0008] (1) Raw material pretreatment;

[0009] (2) Primary enzymatic hydrolysis: Add 0.1 - 1 wt% pepsin, enzymatically hydrolyze at 10°C - 40°C for 1 - 4 h, centrifuge to obtain the supernatant, and adjust the pH to 6 - 8 with 0.1 wt% NaOH solution to obtain the primary enzymatic hydrolysate;

[0010] (3) Extract crude collagen: Add neutral salt to the primary enzymatic hydrolysate, let it stand for precipitation and then centrifuge, and the precipitate is crude collagen;

[0011] (4) Secondary enzymatic hydrolysis: Add water to the crude collagen and add 0.1 - 3 wt% compound protease for secondary enzymatic hydrolysis. The compound protease is trypsin: collagenase: flavor protease = 1:0.5 - 5:0.1 - 1 (mass ratio), enzymatically hydrolyze at 30°C - 60°C for 2 - 8 h, inactivate the enzyme and then centrifuge to obtain the supernatant enzymatic hydrolysate, and hydrolyze the crude collagen into small molecule collagen peptides through secondary enzymatic hydrolysis to obtain the secondary enzymatic hydrolysate;

[0012] (5) Decolorize, filter and spray dry the secondary enzymatic hydrolysate to obtain small molecule squid collagen peptide.

[0013] Further, in the step (1), the squid skin raw material is washed, homogenized after adding water in a ratio of 1:1 - 5, and the pH of the homogenate is adjusted to 1 - 4.

[0014] Further, the neutral salt in the step (3) is one or a mixture of sodium chloride, calcium chloride, ammonium chloride, ammonium sulfate, phosphate, etc. The concentration of the neutral salt reaches 1 - 10 wt%, and salting - out precipitation realizes the purification of collagen and maintains the protein structure and biological activity, and avoids the use of a large amount of acids, bases and organic reagents.

[0015] Further, in the step (4), the trypsin includes trypsin, pancreatic amylase and pancreatic lipase. Trypsin and collagenase have high hydrolysis ability for the hydrolysis of collagen. Pancreatic lipase and pancreatic amylase can decompose fat and starch components, and flavor protease reduces the bitterness caused by hydrolysis. The molecular weight of collagen peptide is controlled through the synergistic hydrolysis effect of the compound enzyme, and the degreasing and debittering of the peptide powder are realized.

[0016] Further, in the step (5), 0.1-1 wt% of adsorbent is added to the secondary enzymolysis solution and stirred for 2 h, and then decolorization is completed by filtration through a filter press, followed by desalting through nanofiltration membrane filtration to obtain a clear, transparent and nearly colorless enzymolysis solution; the adsorbent for decolorization is one or a mixture of several of activated carbon, activated clay, perlite, bentonite, etc.; the secondary enzymolysis solution is filtered successively by a ceramic membrane filtration device with a pressure of 0.4 MPa and a nanofiltration membrane device with a pore size of 200D, and then enters a spray tower for spray drying, with the inlet air temperature for drying being 170-200 °C and the outlet temperature being 85-105 °C.

[0017] Application of the squid collagen peptide obtained by the above preparation method in the preparation of food, health products, medical supplies and cosmetics.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] Most of the commonly used collagen peptide extraction technologies require repeated treatments with acids, alkalis and organic reagents for swelling and degreasing, etc., and are formed by single enzymolysis and drying. The process is not environmentally friendly and has high requirements for equipment. The present invention utilizes two-step enzymolysis, and the process is simple and easy for industrial production.

[0020] In the present invention, collagen is hydrolyzed by an enzymatic method to maintain the collagen structure and avoid the destruction of amino acids. The use of acids, alkalis and organic solvents in the preparation process is reduced, and the loss of equipment and environmental pollution are avoided. The present invention uses the synergistic effect of compound proteases for the second enzymolysis, which can achieve low molecular weight, degreasing and debittering of the peptide powder, and the obtained peptide powder has a low molecular weight, good flavor and no peculiar smell, and is suitable for human consumption and digestion and absorption.

[0021] Moreover, through experimental verification, the squid collagen peptide prepared by the present invention has strong antioxidant ability and has remarkable effects in aspects such as whitening, anti-wrinkle and antioxidant. Description of the Drawings

[0022] Figure 1 It is a representative diagram of the test results of antioxidant efficacy.

[0023] Figure 2 It is a bar chart of the test results of antioxidant efficacy.

[0024] Figure 3 It is a bar chart of the relative expression level of zebrafish elastin gene.

[0025] Figure 4 It is a bar chart of the relative expression level of zebrafish type I collagen gene.

[0026] Figure 5 It is a representative diagram of the test results of whitening efficacy.

[0027] Figure 6It is a bar graph of zebrafish melanin inhibition.

[0028] Figure 7 It is a representative graph of the repair effect test results.

[0029] Figure 8 It is a bar graph of the repair effect test results. Detailed implementation manners

[0030] The technical solution of the present invention will be further described and illustrated below in conjunction with embodiments.

[0031] Reagents, enzyme products, etc. used in the following examples are all commercially available products.

[0032] Example 1

[0033] A preparation method of squid collagen peptide, comprising the following steps:

[0034] (1) Raw material pretreatment: Wash the squid skin raw material, and homogenize the squid skin with water at a ratio of 1:2.

[0035] (2) Primary enzymatic hydrolysis: Adjust the pH of the homogenate to 2.0 with citric acid, add 0.15% pepsin, and hydrolyze at 35°C for 2 h. Centrifuge and take the supernatant, and adjust the pH to 7.0 with 0.1% NaOH solution.

[0036] (3) Crude collagen extraction: Add sodium chloride to the primary enzymatic hydrolysis solution in step (2) to make its concentration reach 10%, let it stand for precipitation and then centrifuge to obtain squid crude collagen.

[0037] (4) Secondary enzymatic hydrolysis: Stir the crude collagen in step (3) with water at a ratio of 1:3, add 1% compound protease for secondary enzymatic hydrolysis, hydrolyze at 50°C for 3 h, inactivate the enzyme at 95°C for 5 min, and centrifuge to take the supernatant enzymatic hydrolysis solution; the compound protease is pancreatin: collagenase: flavor protease = 1:2:0.2.

[0038] (5) Decolorization, filtration, concentration, and spray drying: Add 0.5% activated clay adsorbent to the secondary enzymatic hydrolysis solution in step (4), stir for 2 h, filter through a filter press to complete decolorization, and then filter through a nanofiltration membrane to desalt, obtaining a clear, transparent and nearly colorless enzymatic hydrolysis solution, and obtain squid hydrolyzed collagen peptide powder through spray drying; the inlet air temperature for drying is 185°C, and the outlet temperature is 95°C.

[0039] Example 2:

[0040] A preparation method of squid collagen peptide, comprising the following steps:

[0041] (1) Raw material pretreatment: Wash the squid skin raw material, and homogenize it with water at a ratio of 1:5.

[0042] (2) Primary enzymatic hydrolysis: The homogenate was adjusted to pH 3.0 with citric acid, 0.8% pepsin was added, and enzymatic hydrolysis was carried out at 25 °C for 3.5 h. After centrifugation, the supernatant was adjusted to pH 7.5 with 0.1% NaOH solution;

[0043] (3) Extraction of crude collagen: Ammonium sulfate was added to the primary enzymatic hydrolysis solution in step (2) to make its concentration reach 10%, and after standing and precipitation, centrifugation was carried out to obtain crude squid collagen;

[0044] (4) Secondary enzymatic hydrolysis: The crude collagen in step (3) was stirred with water in a ratio of 1:1, and 2% compound protease was added for secondary enzymatic hydrolysis. Enzymatic hydrolysis was carried out at 45 °C for 6 h, and the enzyme was inactivated at 95 °C for 5 min, and then centrifuged to obtain the supernatant enzymatic hydrolysis solution; The compound protease is pancreatin: collagenase: flavor protease = 1:1:0.5.

[0045] (5) Decolorization, filtration, concentration, and spray drying: 0.8% activated carbon adsorbent was added to the secondary enzymatic hydrolysis solution in step (4) and stirred for 2 h. After filtration through a plate filter press, decolorization was completed, and then desalination was carried out through a nanofiltration membrane to obtain a clear, transparent and nearly colorless enzymatic hydrolysis solution. After spray drying, squid hydrolyzed collagen peptide powder was obtained; The inlet temperature for drying was 190 °C, and the outlet temperature was 85 °C.

[0046] Comparative Example 1

[0047] Compared with Example 1, the difference in Comparative Example 1 is only that: the squid skin homogenate was not subjected to primary enzymatic hydrolysis with pepsin and directly subjected to compound enzyme hydrolysis, and the rest is the same as in Example 1.

[0048] Comparative Example 2

[0049] Compared with Example 1, the difference in Comparative Example 2 is only that: the crude collagen obtained from the primary enzymatic hydrolysis solution was subjected to secondary enzymatic hydrolysis in step (4) using a single pancreatin for enzymatic hydrolysis, and the rest is the same as in Example 1.

[0050] Comparative Example 3

[0051] Compared with Example 1, the difference in Comparative Example 3 is only that: the crude collagen obtained from the primary enzymatic hydrolysis solution was subjected to secondary enzymatic hydrolysis in step (4) using a single collagenase for enzymatic hydrolysis, and the rest is the same as in Example 1.

[0052] Comparative Example 4

[0053] Compared with Example 1, the difference in Comparative Example 5 is only that: the crude collagen obtained from the primary enzymatic hydrolysis solution was subjected to secondary enzymatic hydrolysis in step (4) using 1% neutral protease for enzymatic hydrolysis, and the rest is the same as in Example 1.

[0054] Comparative Example 5

[0055] Compared with Example 1, the difference in Comparative Example 4 is only that: the crude collagen obtained from the primary enzymatic hydrolysate is enzymatically hydrolyzed with 1% papain in step (4) of the secondary enzymatic hydrolysis, and the rest is the same as in Example 1.

[0056] I. The samples obtained in the above Examples 1 and 2 and Comparative Examples 1-5 were subjected to antioxidant property detection. The detection method was the ABTS method for determining the antioxidant property of polypeptides in GB / T 39100-2020, and EC 50 represents the half-clearance amount of free radicals:

[0057] The detection results are shown in Table 1:

[0058] Table 1 Results of the determination of the antioxidant property of squid collagen peptides

[0059]

[0060] As can be seen from Table 1, the samples in Comparative Examples 2-5 were prepared by a single enzymatic hydrolysis process to obtain peptide powder samples. The antioxidant properties of the peptide powders obtained by enzymatic hydrolysis with trypsin and collagenase were relatively high. Therefore, the two were selected for enzymatic hydrolysis after compounding.

[0061] The squid collagen peptides prepared by secondary enzymatic hydrolysis with compound protease in the present invention have good antioxidant properties.

[0062] II. The samples obtained in the above Examples 1 and 2 and Comparative Examples 1-3 were subjected to flavor sensory and physical and chemical index detection. The detection methods were as follows:

[0063] 1. The molecular weight detection was carried out with reference to the method described in Appendix A of GB 31645 National Food Safety Standard Collagen Peptides;

[0064] 2. The detection of hydroxyproline content was carried out with reference to the method described in GB / T 9695.23-2008 Determination of Hydroxyproline Content in Meat and Meat Products;

[0065] 3. The fat detection was carried out with reference to the method described in GB 5009.6 National Food Safety Standard for Fats in Foods;

[0066] The detection results are shown in Table 2:

[0067] Table 2 Results of the determination of the sensory and physical and chemical indexes of squid collagen peptides

[0068]

[0069] As can be seen from Table 1, compared with the samples in the comparative examples, the collagen peptide samples prepared in the present invention have good flavor, 100% of the molecular weight is less than 3000 Da, high hydroxyproline content, good peptide powder purity, low fat content, and obvious defatting effect of the process.

[0070] III. The squid collagen peptide powder sample prepared in Example 1 was subjected to in vivo antioxidant index detection in zebrafish animal experiments. The detection method is as follows:

[0071] (1) Select healthy zebrafish embryos at 2 days post-fertilization.

[0072] (2) Test grouping: blank control group / positive control group / test substance group setting: Randomly select 24 fish embryos into a 96-well plate, with one fish embryo and 0.2 mL of fish embryo culture medium / 0.2 mL of glutathione working solution / 0.2 mL of test substance solution in each well. Place them in an incubator at 28°C ± 1°C and culture until 72 h ± 1 h post-fertilization. Transfer them to a 24-well plate, with 12 fish embryos and 2 mL of reactive oxygen species (ROS) test solution in each well, and then place them in an incubator at 28°C ± 1°C for 2 h ± 1 h.

[0073] (3) Microscopic analysis of samples: Place the fish embryos on their sides. Take pictures of the fish embryos under a fluorescence stereomicroscope according to unified photographing parameters.

[0074] (4) Data and result calculation:

[0075] Calculate the reactive oxygen species (ROS) scavenging rate: Scavenging rate = (B - S) / B × 100%

[0076] In the formula:

[0077] S—the average value of the "average signal intensity" of ROS in the fish embryos in the test substance treatment group;

[0078] B—the average value of the "average signal intensity" of ROS in the fish embryos in the blank control group;

[0079] p < 0.05 indicates a significant difference.

[0080] Test results: From Figure 1 and Figure 2 By comparison, it was found that the squid collagen peptide sample of the present invention had a 40% ROS scavenging rate for zebrafish embryos at a test concentration of 0.29 g / L (p = 0.000000000000000000000070), and the product had a significant antioxidant effect.

[0081] IV. The squid collagen peptide powder sample prepared in Example 1 was subjected to a test on the efficacy of promoting elastin regeneration. The specific steps are as follows:

[0082] (1) Select healthy zebrafish at 6 days post-fertilization.

[0083] (2) Test grouping: Blank control group / Positive control group / Treated substance group Setup: Randomly select 36 zebrafish, evenly distribute them into 24-well plates, set up 3 wells, with each well containing 12 zebrafish and 2.5 mL of fish embryo culture medium / 2.5 mL of acetyl hexapeptide-8 working solution / 2.5 mL of treated substance solution. Place them in an incubator at 28 ± 1 °C and culture until 24 ± 1 h after fertilization. Collect 12 zebrafish from each well into a 1.5 mL tube, remove the solution, add 0.5 mL of RNAlater solution, and store frozen.

[0084] (3) RNA extraction: Remove the RNAlater solution and rinse 3 times with PBS solution. Place on ice bath, remove the PBS solution, and add 500 μL of TRIzol reagent. Homogenize the zebrafish using a pellet pestle and place on ice bath for 10 min. Add 100 μL of chloroform, vortex for 1 min, and place on ice bath for 5 min. Centrifuge the sample for 20 min, transfer the upper layer solution to a 1.5 mL centrifuge tube, add 250 μL of isopropanol, vortex for 1 min, place on ice bath for 10 min, and centrifuge the sample for 20 min. Remove all the supernatant, add 500 μL of ethanol, vortex for 1 min, centrifuge the sample for 5 min, and place on ice bath for 10 min. Repeat this step 3 times. Remove the ethanol, centrifuge the sample for 5 min, and open the lid in the fume hood to air-dry the sample for 10 min. Add 10 μL of DNase / RNase-free ultrapure distilled water and heat at 55 °C for 15 min.

[0085] (4) cDNA synthesis: Dilute the RNA sample to 1000 ng / 7 μL with DNase / RNase-free ultrapure distilled water. Synthesize cDNA from the RNA using the PrimeScript RT reagent kit with gDNA Eraser (Takara; Cat no. RR047A) and store frozen.

[0086] (5) Perform real-time RT-PCR: Prepare primer mixtures and real-time PCR master mixtures for each primer. Dilute the cDNA sample 10-fold with DNase / RNase-free ultrapure distilled water. Add 9 μL of PCR master mixture and 1 μL of diluted cDNA sample to each well of a 96-well plate for PCR. Seal the 96-well plate for PCR with an optical adhesive film, centrifuge the plate for 5 min, and perform real-time PCR amplification.

[0087] (6) Data and result calculation: Collect real-time PCR data. Ct is used as the amplification result, and the amplification amount of the β-actin gene is used as the housekeeping gene to calculate the relative expression level of each gene (Elna) as the test result.

[0088]

[0089] ΔΔCt = ΔCt 空白对照组Average - ΔCt 受试物 ……

[0090] Relative gene expression level = 2 -ΔΔCt ……………………

[0091]

[0092] In the formula:

[0093] ΔCt: The difference between the cycle number of the target gene and the cycle number of the housekeeping gene;

[0094] Ct target gene: The cycle number experienced when the fluorescence signal of the target gene reaches the set threshold;

[0095] The cycle number experienced when the fluorescence signal of the housekeeping gene reaches the set threshold;

[0096] ΔΔCt: The difference between the average value of ΔCt in the blank control group and ΔCt in the test substance group;

[0097] ΔCt blank control group: The average value of the difference between the cycle number of the target gene and the cycle number of the housekeeping gene in the blank control group;

[0098] ΔCt test substance: The difference between the cycle number of the target gene and the cycle number of the housekeeping gene in the test substance group;

[0099] Blank control group: The relative gene expression level of the target gene in the blank control group;

[0100] Test substance group: The relative gene expression level of the target gene in the test substance group.

[0101] p < 0.05 indicates significant difference.

[0102] (7) Test results

[0103] After measurement, as Figure 3 shown, the promotion rate of the squid collagen peptide sample of the present invention on the expression of the Elna gene in zebrafish is 314% (p = 0.00035) at a test concentration of 0.29 g / L. The sample can significantly promote the expression of the Elna gene in zebrafish and has the effect of promoting elastin regeneration.

[0104] V. Perform the test on the efficacy of promoting collagen regeneration on the squid collagen peptide powder sample prepared in Example 1, and the experimental steps are the same as those for the test on the efficacy of promoting elastin regeneration.

[0105] Data and result calculation: Collect real-time PCR data. Ct is used as the amplification result, and the amplification amount of the β-actin gene is used as the housekeeping gene. Calculate the relative expression levels of each gene (col1a1a, col1a1b, and col1a2) as the test results.

[0106] Test results: After measurement, as Figure 4 shown, the promotion rates of the squid collagen peptide sample prepared in the present invention on the expression of col1a1a, col1a1b, and col1a2 genes in zebrafish at a test concentration of 0.29 g / L are 27% (p = 0.010), 49%

[0107] (p = 0.000022), 239% (p = 0.000022), respectively. The sample can significantly promote the expression of type I collagen genes in zebrafish and has the effect of promoting the regeneration of type I collagen.

[0108] V. Test the whitening effect of the squid collagen peptide powder sample prepared in the example. The test steps are as follows:

[0109] (1) Select healthy zebrafish embryos 6 - 8 h after fertilization.

[0110] (2) Test grouping:

[0111] Setting of blank control group / positive control group / test substance group: Randomly select 20 fish embryos into a 96-well plate, with one fish embryo and 0.2 mL of fish embryo culture solution / solvent solution / 0.2 mL of kojic acid solution / 0.2 mL of test substance solution in each well.

[0112] Setting of 100% melanin inhibition model group: Randomly select 20 fish embryos into a 96-well plate, with one fish embryo and 0.2 mL of phenylthiourea working solution in each well.

[0113] Place them in an incubator at 28°C ± 1°C and culture until 48 h ± 1 h after fertilization.

[0114] (3) Microscopic analysis of samples: Randomly select at least 12 fish embryos from each group for testing. Under a microscope (such as a 2x eyepiece), carefully peel the embryo shell of the unhatched fish embryos with forceps without damaging the fish body. Cover the fish embryos with 2% - 4% methylcellulose and place them with their backs up. Then place them under a stereomicroscope for photography. All fish embryos need to be photographed with the same photography parameters.

[0115] (4) Data and result calculation:

[0116] Melanin inhibition rate = (B - S) / (B - P) × 100%

[0117] In the formula:

[0118] S—the average value of the "average signal intensity" of the fish embryos in the test substance treatment group;

[0119] B—the average value of the "average signal intensity" of the fish embryos in the blank control group;

[0120] P—the average value of the "average signal intensity" of the fish embryos in the 100% melanin inhibition model group.

[0121] p < 0.05 indicates a significant difference.

[0122] (5) Test results

[0123] After measurement, as Figure 5 、 Figure 6 shown, the melanin inhibition rate of the squid collagen peptide sample of the present invention is 18% (p = 0.00019) at a test concentration of 0.07 g / L for zebrafish embryos. The sample can significantly inhibit the melanin production of zebrafish embryos and has a whitening effect.

[0124] VI. Test the repair efficacy of the squid collagen peptide powder sample prepared in Example 1. The specific steps are as follows:

[0125] (1) Select healthy zebrafish embryos 3 days after fertilization.

[0126] (2) Injury model: Anesthetize the zebrafish embryos with tricaine solution and excise the caudal fins of the zebrafish embryos with an experimental scalpel under a microscope.

[0127] (3) Test grouping: Blank control group setting: Randomly select 24 uncut normal fish embryos into a 96-well plate, with one fish embryo and 0.2 mL of fish embryo culture medium in each well.

[0128] Model control group / positive control group / test substance group setting: Randomly select 24 caudal fin cut fish embryos into a 96-well plate, with one fish embryo and 0.2 mL of fish embryo culture medium / 0.2 mL of Rehmannia glutinosa extract solution / 0.2 mL of test substance solution in each well.

[0129] Place them in an incubator at 28°C ± 1°C for 48 h ± 1 h.

[0130] (4) Microscopic analysis of the samples: Anesthetize the zebrafish with tricaine and then take a side photo of the caudal fin of the fish embryo under a stereomicroscope.

[0131] (5) Data and result calculation:

[0132] Caudal fin repair promotion rate: Promotion rate = (S - M) / (B - M) × 100%

[0133] In the formula:

[0134] S—the average value of the caudal fin length of the fish embryos in the test substance treatment group;

[0135] M—the average value of the caudal fin length of the fish embryos in the model control group;

[0136] B—the average value of the caudal fin length of the fish embryos in the blank control group;

[0137] p < 0.05 indicates a significant difference.

[0138] (6) Test results

[0139] After measurement, as Figure 7 、 Figure 8 shown, the promotion rate of the squid collagen peptide sample of the present invention for the repair of the caudal fin of zebrafish embryos is 7% (p = 0.020) at a test concentration of 0.035 g / L. The sample can significantly promote the regeneration of the caudal fin of zebrafish embryos and has a promoting repair effect.

[0140] Finally, although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing squid collagen peptide with beauty and skin care effects, characterized in that: The preparation method comprises the following steps: (1) Raw material pretreatment; (2) Primary enzymatic hydrolysis: add 0.1-1wt% pepsin, perform enzymatic hydrolysis at 10℃-40℃ for 1-4h, centrifuge and take the supernatant, adjust the pH to 6-8 with 0.1wt% NaOH solution to obtain a primary enzymatic hydrolyzate; (3) Extracting crude collagen: adding neutral salt to the primary enzymatic hydrolyzate, allowing it to stand for precipitation and then centrifuging to obtain crude collagen; (4) Secondary enzymatic hydrolysis: adding water to the crude collagen and adding 0.1-3wt% of a composite protease for secondary enzymatic hydrolysis, wherein the composite protease is pancreatic enzyme: collagenase: flavor protease = 1:0.5-5:0.1-1, and performing enzymatic hydrolysis at 30°C-60°C for 2-8h. After inactivating the enzyme, centrifuging and taking the supernatant enzymatic hydrolyzate, the crude collagen is hydrolyzed into small molecule collagen peptides by secondary enzymatic hydrolysis to obtain a secondary enzymatic hydrolyzate; (5) The secondary enzymatic decolorization, filtering, and spray drying are performed to obtain small molecule squid collagen peptides.

2. The method for preparing squid collagen peptide according to claim 1, characterized in that: In the step (1), the squid skin material is washed, water is added in a ratio of 1:1-5, and then homogenized, and the pH of the homogenate is adjusted to 1-4.

3. The method for preparing squid collagen peptide according to claim 1, characterized in that: The neutral salt described in step (3) is one or a mixture of sodium chloride, calcium chloride, ammonium chloride, ammonium sulfate, phosphate, etc., and the concentration of the neutral salt is 1-10wt%.

4. The method for preparing squid collagen peptide according to claim 1, characterized in that: In the step (5), 0.1-1wt% adsorbent is added to the secondary enzymatic hydrolysate and stirred for 2h, decolorized after filtration through a filter press, and then desalted through a nanofiltration membrane to obtain a clear, transparent, nearly colorless enzymatic hydrolysate; the decolorized adsorbent is one or a mixture of activated carbon, activated clay, perlite, bentonite, etc.; the secondary enzymatic hydrolysate is filtered in sequence using a ceramic membrane filtration device with a pressure of 0.4MPa and a nanofiltration membrane device with a pore size of 200D, and then fed into a spray tower for spray drying, with a drying inlet air temperature of 170-200°C and an outlet temperature of 85-105°C.

5. Application of the squid collagen peptide prepared by the preparation method of claim 1 in the preparation of food, health products, medical products and cosmetics.

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