High-activity squid skin antioxidant peptide and preparation method thereof

Abstract

The invention relates to a high-activity squid skin antioxidant peptide. The amino acid sequence of the antioxidant peptide is Gln-Asp-Gly-Phe-Gln-Gly-Ala-His or Ser-Gly-Asp-Asn-Leu-Arg-Phe-Pro. The invention also relates to a preparation method of the antioxidant peptide. According to the squid skin antioxidant peptide, the peptide sequence is obtained through enzymolysis, then the squid skin antioxidant peptide sequence which is high in antioxidant activity and free of toxicity is accurately screened from 343 peptide sequences in a molecular docking and database prediction simulation mode, the screening process is simplified, and the experiment cost is greatly saved.

Description

Technical Field

[0001] The invention relates to the field of marine biological protein peptides, in particular to a highly active squid skin antioxidant peptide and a preparation method thereof. Background Art

[0002] As a natural antioxidant, antioxidant peptides have the advantages of low molecular weight, high activity, easy absorption, no side effects, and have attracted widespread attention for their nutritional value and functions such as lowering blood pressure and improving immunity. Antioxidant peptides are widely used in the food field because they meet the characteristics of "natural, safe, non-toxic, and high nutritional value" and have become one of the most commonly used natural products in functional foods. The use of low-cost biological resources to develop nutritious and safe antioxidant peptides has always been a research hotspot in the field of peptides.

[0003] Squid, also known as squid and cuttlefish, belongs to the phylum Mollusca, class Cephalopoda, subclass Coleoptera, order Decapoda, order Sciuridae, and suborder Osmoothia. It is an important marine economic animal and marine food. In 2020, my country's squid catch reached 295,600 tons, and the output is also increasing. In the processing of squid, about 30% of squid by-products will be produced, mainly squid skin, squid ink, squid cartilage, squid viscera, etc. At present, most of the squid processing by-products are not effectively utilized or are discarded, causing serious waste of resources and environmental pollution. Squid skin accounts for about 8% to 13% of the total squid by-products. It is rich in protein, and protein accounts for about 88% of the dry weight of squid skin. The collagen content accounts for about 50% of the squid skin protein, which is a good potential source of bioactive peptides.

[0004] Bioinformatics tools are used to assist in screening active peptides. Based on the known amino acid sequences in active peptides, database searches and software analysis are used to predict the possible biological activity, safety, and bioaccessibility of peptides, select target peptides for chemical synthesis, and verify their related properties. Bioinformatics tools can save a lot of time and experimental costs. The Keap1-Nrf2 pathway can maintain cellular redox balance and metabolism by regulating more than 600 related genes, and is one of the important signaling pathways for the body to cope with oxidative stress. In recent years, homology modeling and molecular docking have been widely used to reveal the binding sites between antioxidant peptides and their receptor Keap1, providing an effective way to screen and discover antioxidant peptides. However, there are no reports on the use of bioinformatics tools to screen squid skin antioxidant peptides. Summary of the invention

[0005] The first technical problem to be solved by the present invention is to provide a high-activity squid-derived antioxidant peptide screened by bioinformatics in view of the current status of the prior art.

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned highly active squid-derived antioxidant peptides in view of the current status of the prior art.

[0007] The third technical problem to be solved by the present invention is to provide a method for screening the above-mentioned highly active squid-derived antioxidant peptides in view of the current status of the prior art.

[0008] The fourth technical problem to be solved by the present invention is to provide an application of the above-mentioned highly active squid-derived antioxidant peptide in view of the current status of the prior art.

[0009] The technical solution adopted by the present invention to solve the first technical problem is: a highly active squid skin antioxidant peptide, the amino acid sequence of the antioxidant peptide is Gln-Asp-Gly-Phe-Gln-Gly-Ala-His or Ser-Gly-Asp-Asn-Leu-Arg-Phe-Pro.

[0010] The technical solution adopted by the present invention to solve the above second technical problem is: a method for preparing the above squid skin antioxidant peptide comprises the following steps:

[0011] (1) Pretreatment of squid skin

[0012] Take the squid skin, grind it, and dry it with gauze to obtain squid skin crumbs;

[0013] (2) Preparation of highly active enzymatic hydrolysates

[0014] The squid skin crumbs obtained in step (1) are prepared into a suspension with a buffer solution, alkaline protease is added for enzymolysis, and after the enzymolysis is completed, the supernatant is collected to obtain an enzymolysis product;

[0015] (3) Separation of highly active enzymatic hydrolysis products

[0016] The enzymatic hydrolysate prepared in step (2) is separated by a gel column, and the antioxidant activity of each separated fraction is measured to obtain the separated fraction with the highest activity.

[0017] Preferably, the pH of the buffer solution in step (2) is 8.0-10.0, and the squid skin crumbs obtained in step (1) are prepared into a suspension with a solid-liquid ratio of 1:1-7 using the buffer solution, 1500-3500 U / g of alkaline protease is added based on the substrate mass, and then enzymolysis is performed at 40-60° C. for 1-5 h to obtain an enzymolysis solution, and finally the enzymolysis solution is kept at 95-100° C. for 15-25 min, cooled, centrifuged at 8000-10000 r / min, and the supernatant is collected, and the supernatant is concentrated and freeze-dried to obtain an enzymolysis product.

[0018] Preferably, the pH of the buffer solution in step (2) is 10, and the squid skin crumbs obtained in step (1) are prepared into a suspension with a solid-liquid ratio of 1:4.5 using the buffer solution, 2625 U / g of alkaline protease based on the substrate mass is added, and then enzymolysis is performed at 40°C for 4.5 hours to obtain an enzymolysis solution, and finally the enzymolysis solution is kept at 100°C for 15 minutes, cooled, centrifuged at 10000 r / min and the supernatant is collected, and the supernatant is concentrated and freeze-dried to obtain an enzymolysis product.

[0019] Preferably, the gel column in step (3) is a Sephadex G-15 gel chromatography column, and the specific conditions for separating and purifying the enzymatic hydrolysis product are: the eluent is pure water, the detection wavelength is 220 nm, the sample amount is 100 mg, and the flow rate is 0.5 mL / min.

[0020] The technical solution adopted by the present invention to solve the third technical problem is: a screening method for highly active squid skin antioxidant peptides, screening the highest activity separation fraction obtained by the above preparation method, the screening method comprising the following steps:

[0021] (4) performing sequence identification on the highest activity isolated fraction to obtain a squid skin peptide sequence group;

[0022] (5) Molecular docking of the squid skin peptide sequence group identified in step (4) with the Keap1 protein to screen out squid skin peptide sequences with high activity.

[0023] Preferably, in step (4), the specific process of sequence identification is: measuring the highest activity separation fraction by LC-MS / MS, and then performing sequence analysis by using the Denovo method.

[0024] Preferably, in step (5), the PDB file 2FLU of the 16mer Nrf2 binding in the Kelch region of the Keap1 protein is used as the docking receptor file, and preprocessing is performed to define the active center coordinates as X=5, Y=9, Z=1, and the active center radius as 9.5A.

[0025] Preferably, in step (5), each sequence in the squid skin peptide sequence group is used as a ligand, and all ligand structures are generated by Chem3D and subjected to energy minimization using the CHARMm force field.

[0026] Preferably, in step (5), the receptor and the ligand are docked by CDDOCK to screen the squid skin peptide sequence with a high Keap1 protein-CDDOCKEnergy value.

[0027] The technical solution adopted by the present invention to solve the fourth technical problem is: an application of the above-mentioned squid skin antioxidant peptide in the preparation of food additives or daily chemical products.

[0028] Compared with the prior art, the advantages of the present invention are:

[0029] (1) The antioxidant peptides screened from squid skin in the present invention have good antioxidant activity and are non-cytotoxic;

[0030] (2) The squid skin antioxidant peptide of the present invention has no toxic side effects and has intestinal absorption ability according to ADMET prediction. The squid skin antioxidant peptide has in vivo biological activity according to Peptide Ranker prediction. Stability experiments show that the activity of the squid skin antioxidant peptide remains above 70% in acidic and hot environments, thus having good application prospects.

[0031] (3) The present invention uses molecular docking and database prediction simulation methods to accurately screen out squid skin antioxidant peptide sequences with high antioxidant activity and no toxicity from 343 peptide sequences, which simplifies the screening process and greatly saves experimental costs;

[0032] (4) The raw material used in the present invention is the processing waste of squid, a marine economic fish, which effectively avoids environmental pollution and improves resource utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the Sephadex G-15 gel separation elution diagram in Example 1 of the present invention;

[0034] Figure 2 for Figure 1 The scavenging effect of different separation fractions on free radicals;

[0035] Figure 3 This is a graph showing the effect of temperature on the antioxidant activity of the separated fraction P4 in Example 1 of the present invention;

[0036] Figure 4 This is a graph showing the effect of pH value on the antioxidant activity of the separated fraction P4 in Example 1 of the present invention;

[0037] Figure 5 This is a diagram showing the molecular docking results of QDGFQGAH and Keap1 receptor in Example 4 of the present invention;

[0038] Figure 6 This is a diagram showing the molecular docking results of SGDNLRFP and Keap1 receptor in Example 4 of the present invention;

[0039] Figure 7 It is the secondary mass spectrum of QDGFQGAH and SGDNLRFP in Example 4 of the present invention;

[0040] Figure 8 This is a diagram showing the scavenging effect of QDGFQGAH and SGDNLRFP on DPPH free radicals in Example 4 of the present invention;

[0041] Fig. 9 This is a diagram showing the effect of QDGFQGAH and SGDNLRFP on HepG2 cell viability and oxidative stress protection in Example 4 of the present invention;

[0042] Fig.10 This is a graph showing the effect of different temperatures and pH values ​​on the antioxidant activity of QDGFQGAH in Example 4 of the present invention;

[0043] Fig.11 This is a graph showing the effect of temperature and pH value on the antioxidant activity of SGDNLRFP in Example 4 of the present invention;

[0044] Fig.12 This is a diagram showing the enzymatic hydrolysis effects of different proteases on squid skin in Example 5 of the present invention;

[0045] Fig.13 This is a graph showing the single factor experimental results of protease hydrolysis in Example 6 of the present invention. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below with reference to the accompanying drawings.

[0047] Example 1

[0048] The preparation method of squid skin antioxidant peptide comprises the following steps:

[0049] (1) Pretreatment of squid skin

[0050] Take the squid skin, grind it, and dry it with gauze to obtain squid skin crumbs;

[0051] (2) Preparation of highly active enzymatic hydrolysates

[0052] The squid skin crumbs obtained in step (1) are prepared into a suspension with a solid-liquid ratio of 1:4.5 using a buffer solution with a pH of 10.0, 2625 U / g alkaline protease is added according to the substrate mass ratio, and enzymolysis is carried out at 40° C. for 4.5 hours, the enzymolysis solution is kept at 100° C. for 15 minutes to inactivate the enzyme, and after cooling, the solution is centrifuged at 10,000 r / min for 15 minutes, the supernatant is collected, and the high-activity enzymolysis product is obtained by concentration and freeze-drying;

[0053] (3) Separation and purification of enzymatic hydrolysis products

[0054] The enzymatic hydrolysate prepared in step (2) was passed through a Sephadex G-15 gel chromatography column, the eluent was pure water, the detection wavelength was 220 nm, the sample load was 100 mg, the flow rate was 0.5 mL / min, and the eluents of each peak were collected. Each peak was named P1 to P7, as shown in FIG. Figure 1 As shown; the eluates of P1 to P7 were concentrated and lyophilized to obtain lyophilized powders of separated fractions, and the scavenging rates of P1 to P7 on ABTS+ and ·DPPH free radicals were determined respectively. The results are shown in Figure 2 As shown in the figure, the P4 component had the highest scavenging rate for ABTS+ and ·DPPH free radicals, so P4 was selected for stability analysis and peptide identification.

[0055] like Figure 3 As shown, the antioxidant activity of the separated fraction P4 prepared by the present invention was tested at different temperatures, and it was kept at 20°C, 40°C, 60°C, 80°C, and 100°C for 1 hour respectively. The results showed that the activity of the separated fraction P4 was maintained above 80%, showing good thermal stability; Figure 4 As shown, the antioxidant activity of the separated fraction P4 prepared by the present invention was tested at different pH values. It was dissolved in solutions with pH values ​​of 4, 5, 6, 7, 8, and 9 and allowed to stand for 1 hour. The results showed that the activity of the separated fraction P4 in a neutral acidic environment was maintained above 90%, showing good acid stability.

[0056] Example 2

[0057] The preparation method of squid skin antioxidant peptide comprises the following steps:

[0058] (1) Pretreatment of squid skin

[0059] Take the squid skin, grind it, and dry it with gauze to obtain squid skin crumbs;

[0060] (2) Preparation of highly active enzymatic hydrolysates

[0061] The squid skin crumbs obtained in step (1) are prepared into a suspension with a solid-liquid ratio of 1:1 using a buffer solution with a pH of 8.0, 1500 U / g alkaline protease is added according to the substrate mass ratio, and enzymolysis is performed at 45° C. for 5 h, the enzymolysis solution is kept at 98° C. for 20 min to inactivate the enzyme, and after cooling, the solution is centrifuged at 9000 r / min for 15 min, the supernatant is collected, and the high-activity enzymolysis product is obtained by concentration and freeze-drying;

[0062] (3) Separation and purification of enzymatic hydrolysis products

[0063] The enzymatic hydrolysate prepared in step (2) was passed through a Sephadex G-15 gel chromatography column, the eluent was pure water, the detection wavelength was 220 nm, the sample amount was 100 mg, the flow rate was 0.5 mL / min, and the eluent of each peak was collected.

[0064] Example 3

[0065] The preparation method of squid skin antioxidant peptide comprises the following steps:

[0066] (1) Pretreatment of squid skin

[0067] Take the squid skin, grind it, and dry it with gauze to obtain squid skin crumbs;

[0068] (2) Preparation of highly active enzymatic hydrolysates

[0069] The squid skin crumbs obtained in step (1) are prepared into a suspension with a solid-liquid ratio of 1:7 using a buffer solution with a pH of 9.0, 3500 U / g alkaline protease is added according to the substrate mass ratio, and enzymolysis is performed at 60° C. for 1 hour, the enzymolysis solution is kept at 95° C. for 25 minutes to inactivate the enzyme, and after cooling, the solution is centrifuged at 8000 r / min for 15 minutes, the supernatant is collected, and the high-activity enzymolysis product is obtained by concentration and freeze-drying;

[0070] (3) Separation and purification of enzymatic hydrolysis products

[0071] The enzymatic hydrolysate prepared in step (2) was passed through a Sephadex G-15 gel chromatography column, the eluent was pure water, the detection wavelength was 220 nm, the sample amount was 100 mg, the flow rate was 0.5 mL / min, and the eluent of each peak was collected.

[0072] Example 4 Screening and identification of peptides with high antioxidant activity

[0073] (1) Sequence analysis of the highly active fraction P4

[0074] The highly active fraction P4 prepared in Example 1 was measured by LC-MS / MS. The sample was subjected to reductive alkylation treatment. The chromatographic column was Acclaim Pep Map RPLC C 18 (1.9 μm, 150 μm×15 cm). Elution conditions: Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile, flow rate 600 nL·min -1 , 5 μL injection, linear gradient elution (B concentration of 4% to 8%, 2 min, 8% to 28% 43 min, 28% to 40% 10 min, 40% to 95% 1 min, 95% 10 min). Mass spectrometry parameters: spray voltage: 2.2 kV, capillary temperature: 270 ° C, MS resolution: 70000 at 400 m / z. A total of 343 sequences were obtained by sequence analysis using the Denovo method.

[0075] (2) Bioinformatics tools assisted screening of squid skin antioxidant peptides

[0076] All ligand structures were generated by Chem3D and minimized using the CHARMm force field. The PDB file 2FLU of the 16mer Nrf2 binding in the Kelch region of Keap1 protein was used as the docking receptor file. The water in the original crystal was removed using DiscoveryStudio 2019 software, and its active center was preprocessed to define (X=5, Y=9, Z=1; radius: 9.2A). The constructed receptor and ligand were docked by CDDOCK. The higher the -CDD OCK Energy, the more stable the binding of the ligand to the receptor. Peptides with higher -CDDOCK Energy values ​​with Keap1 protein were screened.

[0077] By Peptideranker http: / / distilldeep.ucd.ie / PeptideRanker / ) to predict the activity of biological peptides, and those with a score higher than 0.45 were considered to have in vivo activity and were subsequently screened.

[0078] Use Innovagen's online https: / / www.innovagen.com / proteomics-tools ) tool to evaluate the solubility of peptides in water.

[0079] The ADMETlab 2.0 ( https: / / admetmesh.scbdd.com / service / evaluation / index ) Evaluate the intestinal absorption and toxicity-related properties of the peptides in vivo, and select sequences that have no potential for allergy and toxicity and have good intestinal absorption for subsequent synthesis verification.

[0080] Use the AHTPDB database (available from: https: / / crdd.osdd.net / raghava / ahtpdb / ) and BIOPEP-UWM database (available from: https: / / biochemia.uwm.edu.pl / ) were used to evaluate whether they were novel peptides that were not included in the database. The amino acid sequences of each antioxidant peptide and the corresponding docking energy and antioxidant activity prediction score, water solubility, potential allergenicity, potential toxicity and physicochemical properties are shown in Table 1.

[0081] Table 1 Docking scores and property predictions of antioxidant peptides

[0082]

[0083] The molecular docking results of the antioxidant peptide with the Keap1 receptor shown in the amino acid sequence QDGFQGAH are shown in Figure 5 The molecular docking results of the amino acid sequence SGDNLRFP and Keap1 receptor are shown in Figure 6 .like Figure 5 , 6As shown in the figure, through the binding modes and binding sites of the two antioxidant peptides and the receptor protein Keap1, it can be seen that the binding of the antioxidant peptides to the Keap1 subunit is mainly through hydrogen bonding and electrostatic interactions with the receptor amino acid residues. Among them, the amino acid sequence QDGFQGAH can form hydrogen bonds with 8 residues (Arg380, Asn414, Arg415, Ser431, Gly433, Arg483, Ser508, Ser602) on the Keap1 protein, and can also form electrostatic interactions with Arg380 and Arg480, and hydrophobic interactions with Tyr572 and Tyr577. The amino acid sequence SGDNLRFP can form hydrogen bonds with residues Arg380, Asn414, Arg415, Arg483, Ser508, Tyr525, and Gln530 in the Keap1 protein. In addition, SGDNLRFP can also form hydrophobic interactions with Arg415 and Tyr525, and electrostatic interactions with Arg380, Arg480, and Phe478.

[0084] (3) Synthesis of squid skin peptides and verification of their antioxidant activity in vitro

[0085] According to the results of mass spectrometry sequencing, Anhui Guopeptide Biotechnology Co., Ltd. was commissioned to carry out chemical synthesis. The mass spectrometry detection results of the synthesized amino acid sequences QDGFQGAH and SGDNLRFP peptides are as follows Figure 7 As shown. The antioxidant activity of the peptide was verified by DPPH free radical scavenging experiment and hydrogen peroxide-induced HepG2 model. Figure 8 As shown, both peptides screened have good DPPH free radical scavenging ability. Fig. 9 As shown, the CCK-8 analysis results confirmed that both peptides showed no cytotoxic effect on HepG2 cells, and had a protective effect on HepG2 cells induced by hydrogen peroxide, and at 1 mg / mL, the cell viability was increased from 49.8% to more than 80%. Figure 8 , 9 In the figure, QD GFQGAH and SGDNLRFP are abbreviated as Q-8-H and S-8-P, respectively.

[0086] The antioxidant activity of the polypeptides QDGFQGAH and SGDNLRFP isolated and screened by the present invention was tested under different temperature and pH conditions. The test results are as follows: Fig.10 and Fig.11 As shown, the peptides QDGFQGAH and SGDNLRFP still have good antioxidant activity at 25-80°C, and can maintain high activity under acidic and neutral conditions.

[0087] Example 5 Screening of proteases

[0088] Squid skin was used as raw material, the substrate concentration was fixed at 10%, the enzyme amount was 5000U·g-1, and then enzymolysis was carried out for 4 hours under the optimal temperature and pH conditions of each enzyme, including neutral protease (40℃, pH 7.0), alkaline protease (40℃, pH 10.0), acid protease (50℃, pH 4.0), trypsin (40℃, pH 8.0), and papain (40℃, pH 6.0). After the enzymolysis was completed, the enzyme was inactivated with boiling water for 10 minutes, centrifuged, and the supernatant was taken. The DPPH free radical scavenging rate and hydrolysis degree were used as indicators to screen the optimal protease.

[0089] from Fig.12 It can be seen that the enzymatic hydrolysate obtained by alkaline protease hydrolysis has the highest antioxidant activity and hydrolysis degree, which are 54.26±0.19% and 11.58±0.41% respectively. Therefore, alkaline protease is selected as the hydrolase of the present invention.

[0090] Example 6 Optimization of enzymolysis conditions

[0091] (1) Single factor experimental analysis

[0092] Squid skin was used as raw material, alkaline protease as catalyst, and hydrolysis degree as main index. The samples were fixed with the solid-liquid ratio of 1:5 (g / mL), enzymolysis time of 4h, pH 9, and enzyme addition of 2500U / g for single factor experiment. The effects of different enzyme addition amounts (1500, 2000, 2500, 3000, 3500U / g), different enzymolysis times (1, 2, 3, 4, 5h), different solid-liquid ratios (1:1, 1:3, 1:5, 1:7), different pH values ​​(8, 8.5, 9, 9.5, 10) and different enzymolysis times (1, 2, 3, 4, 5h) on the enzymolysis effect of squid skin were investigated. The results are shown in Fig.13 .

[0093] Depend on Fig.13 It can be seen that with the increase of enzyme addition, the hydrolysis degree and antioxidant activity of squid skin collagen hydrolysate first increased and then decreased. When the enzyme addition amount was 2500 U·g -1 When , the degree of hydrolysis of the enzymatic hydrolyzate is the highest, which is 19.23%. Fig.13 It can be seen that with the extension of enzymatic hydrolysis time, the hydrolysis degree of squid skin enzymatic hydrolysate increases, and after 4 hours of hydrolysis, the hydrolysis degree gradually tends to be flat. The antioxidant activity varies with the hydrolysis time. Considering comprehensively, the hydrolysis time of 4 hours is the most suitable. Fig.13 It can be seen that within the experimental temperature range, as the enzymatic hydrolysis temperature increases, the hydrolysis degree of squid skin enzymatic hydrolysate increases accordingly. When the enzymatic hydrolysis temperature is greater than 50°C, the hydrolysis degree and antioxidant activity tend to be flat, and the enzymatic hydrolysis temperature is best at 50°C. Fig.13It can be seen that the degree of hydrolysis of the enzymatic hydrolyzate increases with the increase of pH, and the best effect is achieved when the pH value is 9. Fig.13 It can be seen that with the increase of the solid-liquid ratio, the hydrolysis degree of the enzymatic hydrolyzate and its DPPH free radical scavenging rate first increased and then decreased, and the best result was obtained when the solid-liquid ratio was 1:5.

[0094] (2) Response surface optimization experiment

[0095] According to the results of the single factor test, a three-factor three-level response surface experiment was carried out with enzyme addition amount (A), enzymatic hydrolysis time (B) and solid-liquid ratio (C) as the main factors affecting enzymatic hydrolysis. The experimental results are shown in Table 2.

[0096] Table 2 Experimental design and results

[0097]

[0098] Taking the hydrolysis degree (Y) as the response value, the experimental data were subjected to polynomial fitting regression, and the regression equation was Y = +19.55 + 0.34A + 0.87B - 0.18C - 0.24AB + 0.31AC - 0.33BC - 1.62A 2 -1.39B 2 -0.55C 2 The variance analysis of the regression equation is shown in Table 3. The regression model is extremely significant (P<0.01), the lack of fit value is not significant (P=0.4749>0.05), and the model correlation coefficient R 2 =0.9808, correction factor R Adj 2 =0.9560, indicating that the model is reliable and can better predict the effect of enzymatic hydrolysis conditions on the hydrolysis degree of squid skin collagen peptides. In addition, F(B)>F(A)>F(C), indicating that the enzymatic hydrolysis time has the greatest impact on the enzymatic hydrolysis effect, followed by the enzyme addition amount and the feed-liquid ratio.

[0099] The DPPH free radical scavenging rate was used as the response value, and the data were regressed by polynomial fitting. The regression equation was Y = +68.31 + 0.95A - 0.49B - 0.37C + 0.037AB - 0.010AC + 0.15BC - 4.37A 2 -1.96B 2 -0.44C 2 .

[0100] The variance analysis of the regression equation is shown in Table 4. The regression model is extremely significant (P<0.01), the lack of fit value is not significant (P=0.8536>0.05), and the model correlation coefficient R 2 =0.9931, correction factor R Adj 2=0.9841, indicating that the model is reliable and can better predict the effect of enzymatic hydrolysis conditions on the effect of squid skin collagen in scavenging protein peptide·DPPH free radicals. In addition, F(A)>F(B)>F(C), indicating that the amount of enzyme added has the greatest impact on antioxidant activity, followed by time and feed-liquid ratio.

[0101] Table 3 Analysis of variance of regression model of hydrolysis degree

[0102]

[0103] Note: “**” indicates extremely significant difference (P<0.01); “*” indicates significant difference (P<0.05).

[0104] Table 4 Analysis of variance of regression model for DPPH free radical scavenging rate

[0105]

[0106] Note: “**” indicates extremely significant difference (P<0.01); “*” indicates significant difference (P<0.05).

[0107] (3) Optimal Condition Prediction and Verification

[0108] According to the above analysis, if the degree of hydrolysis is used as an indicator, the optimal process conditions are: enzyme addition amount 2527.5U·g -1 , enzymatic hydrolysis time 4.34h, solid-liquid ratio 1:4.5; if ·DPPH removal rate is used as an indicator, the optimal enzymatic hydrolysis process is: enzyme addition amount 2625U·g -1 , enzymolysis time 4.19h, solid-liquid ratio 1:4.16, comprehensive analysis results, while considering the feasibility of actual operation, the process conditions of enzymolysis were adjusted to: enzyme addition 2625U·g -1 The hydrolysis time was 4.2 h, and the material-liquid ratio was 1:4.5. The experiment was repeated three times under this condition. The yield of the hydrolysate was 74.27±0.43%, and its hydrolysis degree and its DPPH removal effect were 19.03±0.19% and 67.92±0.52%, respectively, which were consistent with the predicted values. This shows that the model has a high accuracy in predicting the squid skin enzymatic hydrolysis process and has certain feasibility.

Claims

1. A highly active squid skin antioxidant peptide, characterized in that: The amino acid sequence of the antioxidant peptide is Gln-Asp-Gly-Phe-Gln-Gly-Ala-His or Ser-Gly-Asp-Asn-Leu-Arg-Phe-Pro.

2. A method for preparing the squid skin antioxidant peptide as claimed in claim 1, characterized in that The following steps are involved: (1) Pretreatment of squid skin Take the squid skin, grind it, and dry it with gauze to obtain squid skin crumbs; (2) Preparation of highly active enzymatic hydrolysates The squid skin crumbs obtained in step (1) are prepared into a suspension with a buffer solution, alkaline protease is added for enzymolysis, and after the enzymolysis is completed, the supernatant is collected to obtain an enzymolysis product; (3) Separation of highly active enzymatic hydrolysis products The enzymatic hydrolysate prepared in step (2) is separated by a gel column, and the antioxidant activity of each separated fraction is measured to obtain the separated fraction with the highest activity.

3. The preparation method according to claim 2, characterized in that: The pH of the buffer solution described in step (2) is 8.0-10.0, and the squid skin crumbs obtained in step (1) are prepared into a suspension with a solid-liquid ratio of 1:1-7 using the buffer solution, 1500-3500 U / g of alkaline protease is added based on the substrate mass, and then enzymolysis is performed at 40-60° C. for 1-5 hours to obtain an enzymolysis solution, and finally the enzymolysis solution is kept at a temperature of 95-100° C. for 15-25 minutes, and after cooling, it is centrifuged at 8000-10000 r / min and the supernatant is collected, and the supernatant is concentrated and freeze-dried to obtain an enzymolysis product.

4. The preparation method according to claim 3, characterized in that: The pH of the buffer solution described in step (2) is 10, and the squid skin crumbs obtained in step (1) are prepared into a suspension with a solid-liquid ratio of 1:4.5 using the buffer solution, 2625 U / g of alkaline protease is added based on the substrate mass, and then enzymolysis is performed at 40° C. for 4.5 hours to obtain an enzymolysis solution, and finally the enzymolysis solution is kept at a temperature of 100° C. for 15 minutes, cooled, centrifuged at 10000 r / min, and the supernatant is collected, and the supernatant is concentrated and freeze-dried to obtain an enzymolysis product.

5. The preparation method according to claim 2, characterized in that: The gel column in step (3) is a Sephadex G-15 gel chromatography column, and the specific conditions for separating and purifying the enzymatic hydrolysis product are: the eluent is pure water, the detection wavelength is 220nm, the sample amount is 100mg, and the flow rate is 0.5mL / min.

6. A method for screening highly active squid skin antioxidant peptides, characterized in that: The highest activity separation fraction obtained by the preparation method according to any one of claims 2 to 5 is screened, and the screening method comprises the following steps: (4) performing sequence identification on the highest activity isolated fraction to obtain a squid skin peptide sequence group; (5) Molecular docking of the squid skin peptide sequence group identified in step (4) with the Keap1 protein to screen out squid skin peptide sequences with high activity.

7. The screening method according to claim 6, characterized in that In step (4), the specific process of sequence identification is: measuring the highest activity separation fraction by LC-MS / MS, and then using the Denovo method for sequence analysis.

8. The screening method according to claim 6, characterized in that In step (5), the PDB file 2FLU of the 16merNrf2 binding in the Kelch region of the Keap1 protein is used as the docking receptor file, and preprocessing is performed to define the coordinates of the active center as X=5, Y=9, Z=1, and the radius of the active center as 9.5A; each sequence in the squid skin peptide sequence group is used as a ligand, and all ligand structures are generated by Chem3D, and the CHARMm force field is used to minimize their energy.

9. The screening method according to claim 8, characterized in that In step (5), the receptor and the ligand are docked by CDDOCK to screen squid skin peptide sequences with high Keap1 protein-CDDOCK Energy values.

10. Use of the squid skin antioxidant peptide according to claim 1 in the preparation of food additives or daily chemical products.

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