Muscle seeking antioxidant peptide as well as preparation method and application thereof

Abstract

The invention relates to muscula seeking antioxidant peptide as well as a preparation method and application thereof, and belongs to the technical field of biology. The preparation method comprises the following steps: preparing enzymatic hydrolysate by using neutral protease as a raw material, carrying out ultrafiltration and gel chromatography separation and purification on the enzymatic hydrolysate, and then carrying out liquid chromatography-tandem mass spectrometry to obtain three antioxidant peptides, and the amino acid sequences of the three antioxidant peptides are KAGIALNDNFVKL, NQPGGQGNNPGQGAPN and LFSYSDTHRH. The preparation method is simple, and the prepared antioxidant peptide has a good scavenging effect on DPPH free radicals and ABTS free radicals. In cell antioxidant research, the content of malondialdehyde MDA in cells can be reduced to different degrees, the activity of SOD antioxidant enzyme can be improved, and the content of cytokines in oxidative damage cells can be effectively inhibited.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an antioxidant peptide of a sargassum sutsugamushi and a preparation method and application thereof. Background Art

[0002] Peptides are molecular polymers between amino acids and proteins. They are better digested and absorbed in the body than free amino acids. While providing nutrients necessary for human growth and development, they are also beneficial to the life activities of biological organisms. Bioactive peptides with antioxidant activity are also called antioxidant peptides. Because they are safe and non-toxic, they are now one of the important sources of antioxidants.

[0003] my country has rich aquatic product resources, and the total output of aquatic products will reach 71 million tons in 2023. Shellfish has attracted much attention because it contains more unsaturated fatty acids and essential amino acids. In addition to fresh consumption and traditional processing, shellfish can also be used in health care products, cosmetics and other fields. The deep processing and utilization of shellfish has become one of the research hotspots. The Xun's muscle clam is mainly distributed in coastal areas of my country. Due to its small size, it is difficult to eat and has been underdeveloped. However, it has the characteristics of low price and no processed products, and is a high-quality resource for the development of active peptides. Therefore, the use of modern biotechnology to develop Xun's muscle clam antioxidant peptides has broad application prospects. Summary of the invention

[0004] The present invention is to solve the above problems and provides a kind of antioxidant peptide of Sarcoptes spheniscus and a preparation method and application thereof.

[0005] The present invention adopts the following technical scheme: a method for preparing antioxidant peptides from Sarcoptes sutsugamushi:

[0006] (1) Enzymatic hydrolysis of clam meat: fresh clam meat is taken, washed and homogenized, neutral protease is added for enzymatic hydrolysis, and after inactivation of the enzyme, centrifugation is performed to remove the precipitate and obtain an enzymatic hydrolyzate;

[0007] (2) Antioxidant peptide separation: The enzymatic hydrolysate is subjected to ultrafiltration separation through an ultrafiltration membrane to collect a polypeptide solution with a molecular weight of 1000-5000 Da;

[0008] (3) Purification of antioxidant peptides: The above polypeptide solution was filtered through a 0.45 μm filter membrane and separated by gel chromatography using Sephadex G-25 as the separation medium. The antioxidant activity of the eluted fractions corresponding to each absorption peak was measured, and the peak fraction with the highest antioxidant activity was selected and freeze-dried to obtain a fraction containing high-purity antioxidant peptides.

[0009] Furthermore, the amount of the neutral protease added is 400-1600 U / g, the pH is adjusted to 5.5-10.5, and the enzymolysis is carried out at 30-60° C. for 120-360 min.

[0010] Furthermore, in step (3), the eluent is ultrapure water with a flow rate of 0.5 mL / min.

[0011] The invention discloses an antioxidant peptide from Sarcoptes sphenanthera, and the amino acid sequence of the peptide is KAGIALNDNFVKL.

[0012] The invention discloses an antioxidant peptide from Sarcoptes sphenanthera, and the amino acid sequence of the peptide is NQPGGQGNNPGQGAPN.

[0013] The invention discloses an antioxidant peptide from Sarcoptes sphenanthera, and the amino acid sequence of the peptide is LFSYSDTHRH.

[0014] The application of antioxidant peptides from the Clam is used in the fields of functional foods, health products, food additives and cosmetics.

[0015] Before molecular docking of the antioxidant peptides from the clam Myna sylvatica with DPPH, the antioxidant activity, hydrophobicity, isoelectric point and toxicity of the active peptides were predicted.

[0016] The advantages of the present invention are specifically as follows:

[0017] (1) The preparation method of the present invention is simple, the steps are easy to operate, and the antioxidant peptides separated and extracted from the flesh of the clam have a high yield, high purity, strong antioxidant activity, and low cytotoxicity. Therefore, the antioxidant can be used as an antioxidant in food to inhibit lipid autooxidation. At the same time, as a natural antioxidant, it has a relatively broad application prospect in the fields of medicines, cosmetics, etc.;

[0018] (2) The antioxidant peptides separated by the present invention are small molecule peptides with a molecular weight between 1200 and 1600, which are easily digested and absorbed by the human body and have good prospects for development and utilization;

[0019] (3) The raw materials of the present invention are easily available and inexpensive, which increases the added value of the clam, provides a basis for the comprehensive utilization of the clam, and has a good promotion prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the scavenging effect of antioxidant peptides on DPPH free radicals and ABTS free radicals at different enzymatic hydrolysis times in Example 1.

[0021] Figure 2 This is a graph showing the scavenging effect of antioxidant peptides from different ultrafiltration fractions in Example 1 on DPPH free radicals.

[0022] Figure 3 This is a diagram of the gel column separation products of the present invention: peak I component, peak II component, peak III component, peak IV component and their scavenging effect on DPPH free radicals.

[0023] Figure 4The effects of gel column separation products Ⅰ, Ⅱ, Ⅲ, and Ⅳ on HepG2 cell cytotoxicity at different concentrations.

[0024] Figure 5 The effects of three antioxidant peptides at different concentrations on the cytotoxicity of HepG2 cells.

[0025] Figure 6 This is a diagram showing the protective effect of the antioxidant peptide of the present invention on H2O2 oxidative damage in HepG2 cells.

[0026] Figure 7 Graph showing the effects of the three antioxidant peptides (P1, P2, and P3) of the present invention on MDA production and SOD activity in HepG2 cells.

[0027] Figure 8 This is a graph showing the effects of the three antioxidant peptides (P1, P2, and P3) of the present invention on interleukin in HepG2 cells.

[0028] Fig. 9 This is a diagram showing the molecular docking results of the antioxidant peptide (P1) of the present invention and DPPH.

[0029] Fig.10 This is a diagram showing the molecular docking results of the antioxidant peptide (P2) of the present invention and DPPH.

[0030] Fig.11 This is a diagram showing the molecular docking results of the antioxidant peptide (P3) of the present invention and DPPH. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation modes of the present invention are further described below in conjunction with specific drawings.

[0032] Embodiment 1:

[0033] (1) Weigh 5 g of C. sutsuga muscle clam meat and add 15 g of deionized water to homogenize, add neutral protease at an addition amount of 1000 U / g after homogenization, adjust the pH to 6.8, heat to 50° C., and perform enzymolysis for 20 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively, and then centrifuge at 8000 rpm for 15 min, collect the supernatant and determine the scavenging rate of DPPH free radical and ABTS free radical;

[0034] (2) using an ultrafiltration membrane with a molecular weight cutoff range of 10000Da, 5000Da and 1000Da to separate the enzymatic hydrolysate by ultrafiltration, and dividing it into four components with different molecular weight ranges, namely, a component with a molecular weight greater than 10000Da, a component with a molecular weight between 5000Da and 10000Da, a component with a molecular weight between 1000Da and 5000Da, and a component with a molecular weight less than 1000Da, and evaluating the DPPH free radical scavenging rate as an indicator; Figure 3 It can be seen that the components with molecular weight between 1000Da and 5000Da have the best antioxidant activity;

[0035] (3) 1-5KDa molecular weight fractionated samples were collected, filtered through a 0.45μm filter membrane, and separated using a Sephadex G-25 gel chromatography column. The eluent was ultrapure water at a flow rate of 0.5mL / min. The antioxidant activity of the eluted components corresponding to each absorption peak was determined, and four peaks were separated and purified. The components corresponding to the four peaks (peak I component, peak II component, peak III component, peak IV component) were subjected to in vitro antioxidant activity determination. The peak component with the highest antioxidant activity was selected for liquid chromatography-tandem mass spectrometry analysis, and three polypeptides with high antioxidant activity (P1, P2, P3) were obtained.

[0036] Embodiment 2:

[0037] The amino acid sequence of the antioxidant peptide prepared in Example 1 was determined: Peak IV purified from the elution component corresponding to the absorption peak was collected, and the amino acid sequence was determined by LC-MS / MS. The physicochemical properties of the three antioxidant peptides from the sargassum were predicted online using Pedpraw, including hydrophobicity and isoelectric point. The activity of the antioxidant peptides from the sargassum was predicted online using Pepti de Ranker. The toxicity of the antioxidant peptides from the sargassum was predicted online using Tox inPred. The results are shown in Table 1.

[0038] Table 1 Antioxidant peptide amino acid sequences

[0039]

[0040] As shown in Table 1, the three antioxidative peptides of the C. sylvatica are all small molecule peptides with a molecular weight of about 1200-1600 Da. Small molecule peptides are more easily absorbed by the human body, and are non-toxic and have good antioxidant activity after testing. This shows that the antioxidative peptides of the C. sylvatica have good application prospects.

[0041] Embodiment 3:

[0042] 1. Determination of antioxidant activity:

[0043] (1) Determination of DPPH free radical scavenging ability

[0044] The DPPH free radical scavenging rate was determined according to the method of (Liu D, Chen X, Huang J, et al. Generation of bioactive peptides from duck meat during post-mortem aging [J]. Food Chemistry, 2017, 237: 408.) and calculated according to formula (1-1):

[0045]

[0046] A b ——Absorbance value of the mixed solution of the working solution and the solvent of the sample to be tested

[0047] A s ——Absorbance value of the mixed solution of working solution and sample to be tested

[0048] A C ——Absorbance value of the mixed solution of the sample to be tested and anhydrous ethanol

[0049] (2) Determination of ABTS free radical scavenging ability.

[0050] The ABTS·+ free radical scavenging rate was determined by referring to the method of (Siddhuraju P, Becker K. The antiox idant and free radical scavenging activities of processed cowpea (Vigna unguiculata (L.) Walp.) seed extracts [J]. Food Chemistry, 2007, 101 (1): 10-19.) and calculated according to formula (1-2):

[0051]

[0052] A b ——Absorbance value of the mixed solution of the working solution and the solvent of the sample to be tested

[0053] A s ——Absorbance value of the mixed solution of working solution and sample to be tested.

[0054] 2. Determination of cytotoxicity of antioxidant peptides:

[0055] Cytotoxicity was reflected by cell viability. HepG2 cells (1×10 4cels / well). After culturing for 24 hours, the supernatant was removed, the cells were washed with PBS, and the culture medium containing antioxidant peptides (concentrations of 50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL, and 250μg / mL) was added for 12 hours. The supernatant was removed and washed twice with PBS. The culture medium containing CCK-8 was added and cultured for another 2 hours. The absorbance at 450nm was measured. The cell survival rate was calculated by formula (1-3):

[0056]

[0057] A b , A s , A c ——Respectively, the absorbance values ​​of the control group, experimental group and blank group at 450nm;

[0058] 3. Protective effect of antioxidant peptides on cell oxidative damage model:

[0059] The above three antioxidant peptides were selected to study the protective effect on H2O2 oxidative damage of human liver cancer HepG2 cells. The protective effect of synthetic peptides on cells was evaluated by CCK-8 method, and the effects of the three synthetic peptides on superoxide dismutase SOD, malondialdehyde MDA production, and interleukin (IL-2, IL-4, IL-6, IL-10) content in the cell supernatant of HepG2 cells damaged by H2O2 were analyzed.

[0060] 4. Molecular docking of antioxidant peptides with DPPH and protein receptor keap 1:

[0061] The above three antioxidant peptides were selected to study the molecular docking results with DPPH. The three-dimensional structure of the DPPH (CID: 2735032) ligand was obtained from the PubChem database, and Autodock was used for docking, and the docking conformation with the minimum docking energy was selected. Pymol software was used for visualization analysis to obtain the interaction points, and Ligplot software was used to analyze the interaction force.

[0062] Depend on Figure 1 It can be seen from a that the antioxidant activity of the enzymatic hydrolysis stock solution of antioxidant peptides varies with the enzymatic hydrolysis time, and the DPPH free radical scavenging rate is 54.17% to 86.38%.

[0063] Depend on Figure 1 b It can be seen that the ABTS free radical scavenging rate after the original solution was diluted 10 times was 46.47% to 67.18%.

[0064] It can be seen that the optimal time for enzymolysis in the present invention is 240 min.

[0065] Depend on Figure 2As shown in the figure, the DPPH free radical scavenging rate EC of the neutral protease hydrolysis system at a molecular weight of 1000-5000Da 50 It is 0.202~0.449mg / mL.

[0066] Figure 3 a and Figure 3 b It can be seen that the four products obtained by gel column separation (the four peak components appear in sequence: peak I component 30-90min, peak II component: 90-135min, peak III component 140-180min, peak IV component 195-240min) were screened by the scavenging DPPH free radical ability and cell survival rate of the four products. Figure 3 (b) It can be seen that the DPPH clearance rate EC of peak IV 50 The cytotoxicity test of the four component peaks showed that peaks I and II produced toxicity to HepG2 cells at high doses. Figure 4 , the cell viability at the addition amount of 250μg / mL was 72.47% and 88.58% respectively. The cell viability of the components corresponding to peaks III and IV was greater than 98%, and they were non-toxic to HepG2 cells. Therefore, the peak IV components were analyzed by liquid chromatography tandem mass spectrometry to obtain the amino acid structures of three polypeptides (P1, P2, P3, amino acid sequences are SEQ.ID.NO.1-3).

[0067] Depend on Figure 5 It can be seen that the three antioxidant peptides at different concentrations have cytotoxic effects on HepG2 cells. At a concentration of 200 μg / mL, P3 has the greatest cytotoxicity.

[0068] Depend on Figure 6 It can be seen that the three antioxidant peptides (P1, P2, and P3) have a protective effect on H2O2 oxidative damage in HepG2 cells.

[0069] Depend on Figure 7 It can be seen that in the cell supernatant of HepG2 cells treated with three antioxidant peptides (P1, P2, P3) when the cells were damaged by H2O2, the production of malondialdehyde (MDA) decreased and the superoxide dismutase (SOD) increased, which indicates that the three antioxidant peptides (P1, P2, P3) have good antioxidant properties.

[0070] Depend on Figure 8 a. Figure 8 b. Figure 8 c and Figure 8It can be seen that the three antioxidant peptides (P1, P2, and P3) obtained by the present invention have a protective effect on H2O2-induced oxidative damage of HepG2 cells, can improve the survival rate of HepG2 cells damaged by H2O2, reduce the amount of MDA generated in cells, increase the activity of cell SOD, and effectively inhibit the content of cytokines (IL-2, IL-4, IL-6, and IL-10) in oxidatively damaged cells.

[0071] Depend on Figure 9-11 It can be seen that the key amino acids in the active site of P1 (KAGIALNDNFVKL) are Val-11, Lys-12 and Leu-13, the key amino acids in the active site of P2 (NQPGGQGNNPGQGAPN) are Asn-8 and Gly-11, and the key amino acids in the active site of P3 (LFSYSDTHRH) are Ser-3 and Ser-5. P1 does not form a hydrogen bond with DPPH, but Phe-10, Val-11, Lys-12, and Leu-13 residues form hydrophobic interactions with DPPH; the Gly-11 residue of P2 forms a hydrogen bond with DPPH, and the Gly-7, Asn-8, Asn-9, and Pro-10 residues form hydrophobic interactions with DPPH; the Ser-3 residue of P3 forms a hydrogen bond with DPPH, and the Tyr-4, Ser-5, and Thr-7 residues form hydrophobic interactions with DPPH.

[0072] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing an antioxidant peptide from Myna sutsugamushi, characterized in that: The steps include: (1) Enzymatic hydrolysis of clam meat: fresh clam meat is washed and homogenized, neutral protease is added for enzymatic hydrolysis, and after inactivation of the enzyme, centrifugation is performed to remove the precipitate and obtain an enzymatic hydrolyzate; (2) Antioxidant peptide separation: The enzymatic hydrolysate is separated by ultrafiltration to obtain a polypeptide solution with a molecular weight of 1000-5000 Da; (3) Purification of antioxidant peptides: The above polypeptide solution was filtered through a 0.45 μm filter membrane and then subjected to gel chromatography separation using Sephadex G-25 as the separation medium. The antioxidant activity of the eluted fractions corresponding to each absorption peak was determined. The peak fraction with the highest antioxidant activity was selected and freeze-dried to obtain a fraction containing high-purity antioxidant peptides. The antioxidant peptides were sequenced.

2. The method for preparing the antioxidant peptide of Clam sutchuenensis according to claim 1, wherein: In the step (1), the amount of neutral protease added is 400-1600 U / g, the pH is adjusted to 5.5-10.5, and the enzymolysis is carried out at a temperature of 30-60° C. for 120-360 minutes.

3. The method for preparing the antioxidant peptide of Myna sutsugamushi according to claim 1, characterized in that: In the step (3), the eluent for gel chromatography separation is ultrapure water, and the flow rate is 0.5 mL / min.

4. The antioxidant peptide obtained according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the antioxidant peptide is KAGIALNDNFVKL.

5. The antioxidant peptide obtained according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the antioxidant peptide is NQPGGQGNNPGQGAPN.

6. The antioxidant peptide obtained according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the antioxidant peptide is LFSYSDTHRH.

7. An application of an antioxidant peptide from the clam, characterized in that: Used in functional foods, health products, food additives and cosmetics.

Images