Preparation method of suaeda salsa oligopeptide and determination method of sequence composition of suaeda salsa oligopeptide

Abstract

The invention relates to the technical field of processing and application of bioactive peptides, and particularly discloses a preparation method of suaeda salsa oligopeptide and a determination method of sequence composition of the suaeda salsa oligopeptide, the molecular weight of the suaeda salsa oligopeptide is less than 2KDa, and the suaeda salsa oligopeptide contains 48 peptide fragment sequences and has antioxidant activity and in-vitro lipid lowering effect; crude protein is extracted from suaeda salsa and separated to obtain suaeda salsa oligopeptide, the sequence composition of the suaeda salsa oligopeptide is analyzed and determined, and the antioxidant activity and the in-vitro lipid-lowering effect of the suaeda salsa oligopeptide are detected. Compared with the suaeda salsa crude protein, the oligopeptide has good biological activity, is easier to absorb and utilize, and can be widely applied to the industries of medicines, special medical foods, health care products and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of processing and application of bioactive peptides, and particularly relates to a preparation method of Suaeda salsa short peptides and a determination method of sequence composition of the Suaeda salsa short peptides. BACKGROUND

[0002] Peptides are compounds formed by condensation of amino and carboxyl groups provided by two or more amino acids, and are physiological active substances capable of participating in completing physiological activities of a body. Peptides can deliver various trace elements and vitamins and other nutrients to various tissues of the body. Peptides can also participate in cell synthesis and have some special physiological activities and characteristics that proteins and amino acids do not have. The molecular weight of peptides is smaller than that of proteins, and peptides can be divided into short peptides, oligopeptides and polypeptides.

[0003] Bioactive peptides are peptide compounds composed of 20 kinds of amino acids and beneficial or having physiological effects on life activities of a biological body. Generally, bioactive peptides are short peptides composed of 2-10 amino acids, and some are polypeptides composed of 10-50 amino acids, and have effects of antioxidant, antibacterial, blood pressure reduction and the like. In recent years, plant source proteins and animal source proteins have become the main sources for preparing bioactive peptides due to advantages of low price, various types, easy production and safe products, and have gradually stepped into the research field of low-carbon production. It has been found that proteins are decomposed into short peptides under the action of enzymes, and the speed of absorption and metabolism is faster, so bioactive short peptides have broader development prospects.

[0004] Suaeda salsa is a plant with medicinal and edible properties, mainly containing various effective active ingredients such as proteins, dietary fibers, polysaccharides, pigments and flavones, and has various biological activities such as enhancing immunity, anti-inflammatory, antioxidant, reducing sugar and reducing lipid. The seedlings of Suaeda salsa can be used for cooking, and the seeds can be pressed for oil, and the oil contains a high proportion of unsaturated fatty acids.

[0005] Studies have shown that the protein content in fresh and tender stems and leaves of Suaeda salsa is as high as 40% of dry matter, and the content of essential amino acids is very close to the complete protein index, so Suaeda salsa is a high-quality plant protein. Therefore, it is of great value to study the preparation method of Suaeda salsa short peptides, the composition of amino acid sequences contained therein, the antioxidant activity and the in-vitro lipid-lowering effect, and to lay a foundation for the development and utilization of Suaeda salsa.

[0006] To this end, the present application provides a preparation method of Suaeda salsa short peptides and a determination method of sequence composition of the Suaeda salsa short peptides, to solve the above problems. SUMMARY

[0007] The present application aims to provide a preparation method of Suaeda salsa short peptides and a determination method of sequence composition of the Suaeda salsa short peptides, to solve the problems raised in the background.

[0008] To achieve the above object, the present application provides the following technical solutions.

[0009] A preparation method of a short peptide of Suaeda salsa, the short peptide of Suaeda salsa has a molecular weight less than 2KDa, contains 48 peptide segment sequences, and has antioxidant activity and in-vitro lipid-lowering effect.

[0010] Preferably, the preparation method comprises the following steps:

[0011] S1, collecting Suaeda salsa leaves, washing, drying, crushing, sieving (140 meshes) to obtain Suaeda salsa powder;

[0012] S2, adding petroleum ether to the Suaeda salsa powder, mixing thoroughly, standing, filtering, and discarding the filtrate to obtain Suaeda salsa filter residue;

[0013] S3, weighing the Suaeda salsa filter residue obtained in step S2, adding 0.04 mol / L phosphate buffer solution at a ratio of 1:13 (w / v), ultrasonic extraction, centrifuging the obtained extract at 4 DEG C for 15 min at a speed of 6000 r / min, taking the supernatant, adding (NH4)2SO4 powder, standing at 4 DEG C until the saturation reaches 80%, centrifuging at 4 DEG C for 15 min at a speed of 6000 r / min after standing, dissolving the precipitate with PBS buffer solution, placing it in a dialysis bag for dialysis for 24 h, freeze-drying to obtain Suaeda salsa crude protein, and storing at low temperature for standby use;

[0014] S4, configuring the Suaeda salsa crude protein powder into 15 portions of Suaeda salsa protein aqueous solution with the same concentration, performing single-factor experiment on each 5 portions, setting three factors of enzyme addition amount, enzyme hydrolysis temperature and enzyme hydrolysis time, and setting five levels for each factor, namely, the enzyme addition amount is 4000, 6000, 8000, 10000 and 12000 U / g respectively, the enzyme hydrolysis temperature is 45, 50, 55, 60 and 65 DEG C respectively, and the enzyme hydrolysis time is 1, 2, 3, 4 and 5 h respectively, to obtain corresponding extract;

[0015] S5, centrifuging the extract obtained in step S4 to obtain upper clear liquid, and freeze-drying to obtain Suaeda salsa peptide powder;

[0016] S6, according to the Box-Benhnken experimental design principle, taking the yield of Suaeda salsa short peptide as a response value, selecting three factors of enzyme addition amount, enzyme hydrolysis temperature and enzyme hydrolysis time to design response surface experiment, and optimizing the process parameters of enzyme hydrolysis extraction of Suaeda salsa peptide.

[0017] Preferably, the enzyme addition amount is 7200 U / g, the pH value is 8.5, the enzyme hydrolysis temperature is 62 DEG C, the enzyme hydrolysis time is 2 h, and the yield of Suaeda salsa short peptide is 52.33%.

[0018] A determination method of a short peptide sequence of Suaeda salsa, comprising the following steps:

[0019] A1, ultrafiltration: the Suaeda salsa peptide solution is filtered through an ultrafiltration membrane with a molecular weight cut-off of 10, 5 and KDa, macromolecular fragments are removed, the filtrate with a molecular weight less than 3KDa is taken, concentrated, freeze-dried, and Suaeda salsa peptide powder is obtained;

[0020] A2, column chromatography: the Suaeda salsa peptide powder obtained in step A1 is prepared into a sample solution of 3mg / mL with ultrapure water, the treated Sephadex G-25 filler is loaded into a glass chromatography column with a size of 1.6cm*150cm, and column chromatography separation is carried out; the sample loading amount is 3mL, ultrapure water is used for elution at a flow rate of 20mL / h, and an ultraviolet detector is used for detection; a total of 6 elution peaks are obtained, which are collected and freeze-dried, the antioxidant activity and in-vitro lipid-lowering effect of each component are determined, the component with the highest activity is taken, and low-temperature preservation is carried out for standby use;

[0021] A3, sequence identification: the sample obtained in step A2 is separated by RP-HPLC and then subjected to mass spectrometry analysis by using a Q-Exactive mass spectrometer (Thermo Fisher); the analysis conditions are as follows: detection mode, positive ion; mass-to-charge ratio (m / z) of polypeptide and polypeptide fragment acquisition method, 10 fragment maps (MS2 scan) are acquired after each full scan; analysis time, 60min.

[0022] Compared with the prior art, the present application has the following advantages:

[0023] (1) The Suaeda salsa short peptide prepared by the present application has a molecular weight less than 2KDa and is easy to be absorbed in vivo. The in-vitro antioxidant experimental results show that the Suaeda salsa short peptide has scavenging effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydroxyl free radical (·OH); and the scavenging effects have a dose-dependent relationship. When the concentration of the Suaeda salsa short peptide is 0.25, 0.4 and 0.4mg / mL, the scavenging rates of DPPH, ABTS and ·OH have reached 50%, which shows that the Suaeda salsa short peptide has good antioxidant capacity.

[0024] (2) The in-vitro lipid-lowering experiment research result of the present application shows that in the lipid peroxidation HepG2 cell model induced by oleic acid (OA), the salt marsh short peptide prepared by the present application can dose-dependently inhibit the increase of glutamic pyruvic transaminase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (γ-GT), lactic dehydrogenase (LDH), triglyceride (TG), total cholesterol (TC) and other lipid metabolism related indexes; meanwhile, the content of intracellular superoxide dismutase (SOD) is increased. Therefore, the salt marsh short peptide prepared by the present application can be used for reducing the lipid accumulation of HepG2 cells caused by oleic acid (OA) stimulation. The salt marsh can be used for lipid-lowering effect research, and the short peptide component contained therein may be the main pharmacodynamic material basis. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The figure for the influence of enzyme addition amount on the extraction rate of salt marsh short peptide;

[0026] Figure 2 The figure for the influence of enzyme hydrolysis temperature on the extraction rate of salt marsh short peptide;

[0027] Figure 3 The figure for the influence of enzyme hydrolysis time on the extraction rate of salt marsh short peptide;

[0028] Figure 4 The three-dimensional surface graph for the interaction of enzyme addition amount and enzyme hydrolysis time on the extraction rate of salt marsh short peptide;

[0029] Figure 5 The three-dimensional surface graph for the interaction of enzyme addition amount and enzyme hydrolysis temperature on the extraction rate of salt marsh short peptide;

[0030] Figure 6 The three-dimensional surface graph for the interaction of enzyme hydrolysis time and enzyme hydrolysis temperature on the extraction rate of salt marsh short peptide;

[0031] Figure 7 The column chromatogram of salt marsh peptide liquid;

[0032] Figure 8 The extraction flow chart of salt marsh short peptide;

[0033] Figure 9 The in-vitro antioxidant activity graph of salt marsh short peptide;

[0034] Figure 10 The graph for the influence of different concentrations of salt marsh short peptide on cell survival rate;

[0035] Figure 11 Figure for the effect of different concentrations of short peptides of Suaeda salsa on the lipid metabolism of HepG2 cells;

[0036] Figure 12 Figure for the effect of different concentrations of short peptides of Suaeda salsa on the content of ALT in HepG2 cells;

[0037] Figure 13 Figure for the effect of different concentrations of short peptides of Suaeda salsa on the content of AST in HepG2 cells;

[0038] Figure 14 Figure for the effect of different concentrations of short peptides of Suaeda salsa on the content of LDH in HepG2 cells;

[0039] Figure 15 Figure for the effect of different concentrations of short peptides of Suaeda salsa on the content of SOD in HepG2 cells;

[0040] Figure 16 Figure for the effect of different concentrations of short peptides of Suaeda salsa on the content of TC in HepG2 cells;

[0041] Figure 17 Figure for the effect of different concentrations of short peptides of Suaeda salsa on the content of TG in HepG2 cells;

[0042] Figure 18 Figure for the effect of different concentrations of short peptides of Suaeda salsa on the content of γ-GT in HepG2 cells. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0044] Embodiment 1:

[0045] As Figures 1 to 7 A preparation method of short peptides of Suaeda salsa, which is implemented by the following method:

[0046] Five portions of 2% aqueous solution of Suaeda salsa protein are prepared, and hydrolysis is performed by using alkaline protease, the pH is adjusted to 8.5, the enzyme hydrolysis temperature is 50°C, the enzyme addition amount is 4000, 6000, 8000, 10000 and 12000 U / g respectively, the enzyme hydrolysis time is 2 h, and the short peptide yield is determined.

[0047] Embodiment 2:

[0048] Configured 2% of the halophyte protein aqueous solution five, with alkaline protease hydrolysis, pH adjusted to 8.5, enzyme hydrolysis temperature is 45, 50, 55, 60, 65 ℃, enzyme addition amount is 10000 U / g, enzyme hydrolysis time is 2h, the yield of short peptide is determined.

[0049] Example 3:

[0050] Configured 2% of the halophyte protein aqueous solution five, with alkaline protease hydrolysis, pH adjusted to 8.5, enzyme hydrolysis temperature is 55 ℃, enzyme addition amount is 10000 U / g, enzyme hydrolysis time is 1, 2, 3, 4, 5h, the yield of short peptide is determined.

[0051] Example 1, 2 and 3 respectively study the enzyme addition amount, enzyme hydrolysis time and enzyme hydrolysis temperature three factors on the influence of halophyte short peptide extraction rate.

[0052] Response surface experiment result analysis:

[0053] On the basis of single factor experiment, according to Box-Benhnken experimental design principle, with halophyte short peptide yield as response value, enzyme addition amount, enzyme hydrolysis time and enzyme hydrolysis temperature three factors are designed response value, the corresponding conditions of halophyte short peptide yield at the maximum response value are obtained: enzyme addition amount is 7246.31 U / g, enzyme hydrolysis time is 2h, enzyme hydrolysis temperature is 61.92 ℃, considering the actual operation simplicity, the process parameters are adjusted as: enzyme addition amount is 7200 U / g, enzyme hydrolysis time is 2h, enzyme hydrolysis temperature is 62 ℃, the predicted value of short peptide yield under this condition is 52.96%.

[0054] In order to verify the result of response surface optimization method, the above optimization extraction condition is used for 3 repeated experiments, and the halophyte short peptide yield is (52.33+0.50)%, which is close to the predicted value, so the model can better reflect the preparation conditions of halophyte short peptide, and the optimization method is feasible.

[0055] The enzyme hydrolysis method is used for extracting halophyte short peptide in the application, the optimal process conditions are determined through single factor and response surface optimization experiments: enzyme addition amount is 7200 U / g, enzyme hydrolysis time is 2h, enzyme hydrolysis temperature is 62 ℃, and the halophyte short peptide yield is 52.33%.

[0056] Experiments 1-3 study the influence of enzyme addition amount, enzyme hydrolysis time and enzyme hydrolysis temperature three single factors on the extraction rate of halophyte short peptide (as shown in Figures 1 to 7 ).

[0057] Example 4:

[0058] As shown in Figure 8 , a method for determining the sequence composition of halophyte short peptide comprises:

[0059] The salt desert halophyte peptide solution was passed through ultrafiltration membranes with molecular weight cutoffs of 10, 5, and 3 KDa to remove macromolecular fragments, and the filtrate less than 3 KDa was concentrated and lyophilized to obtain a salt desert halophyte short peptide sample.

[0060] The treated Sephadex G-25 filler was loaded into a 1.6 cm x 150 cm glass chromatography column, and a salt desert halophyte short peptide sample was prepared into a 3 mg / mL solution with ultrapure water, with a sample loading amount of 3 mL. The sample was eluted with ultrapure water at a flow rate of 20 mL / h, while being detected with a UV detector. The elution peak was collected, freeze-dried, and the antioxidant activity of each component was determined. The component with the highest antioxidant activity was selected and stored at low temperature for future use.

[0061] The sample obtained above was separated by RP-HPLC and subjected to mass spectrometry analysis using a Q-Exactive mass spectrometer (Thermo Fisher). The analysis conditions were as follows: detection mode, positive ion; mass-to-charge ratio acquisition method for polypeptides and fragments of polypeptides, 10 fragment spectra (MS 2 scans) collected after each full scan; analysis duration, 60 min.

[0062] Example 5:

[0063] As Figure 9 shown, the in vitro antioxidant activity of a salt desert halophyte short peptide was studied using the following method:

[0064] 1) DPPH free radical scavenging experiment

[0065] A certain amount of salt desert halophyte short peptide was dissolved, 1 mL of sample solution was taken, 4 mL of DPPH solution with a concentration of 0.1 mmol / L was added, and the mixture was mixed uniformly. After 30 min of reaction in the dark, the absorbance value was measured at a detection wavelength of 595 nm. Each group was measured in triplicate, and the relationship curve between DPPH free radical scavenging rate (Y) and sample concentration (X) was drawn.

[0066] 2) ABTS free radical scavenging experiment:

[0067] ABTS and potassium sulfite at a concentration of 7 mmol / L and 4.8 mmol / L, respectively, were mixed in equal amounts and placed in the dark at room temperature for 12-16 h to obtain an ABTS free radical stock solution. The ABTS free radical stock solution was diluted to an absorbance value of (0.700 ± 0.001) for standby use.

[0068] Different mass concentrations of salt desert halophyte short peptide solution 0.2 mL and 3.8 mL of ABTS solution were mixed uniformly and placed at room temperature for 6 min. The absorbance was measured at 734 nm, and each group was measured in triplicate. The relationship curve between ABTS free radical scavenging rate (Y) and sample concentration (X) was drawn.

[0069] 3)·OH radical scavenging experiment:

[0070] The Fe2SO4 solution with a concentration of 9 mmol / L, the ethyl salicylate solution with a concentration of 9 mmol / L and the H2O2 solution with a concentration of 8.8 mmol / L were configured. 1 mL of the Suaeda salsa short peptide solution with different mass concentrations was measured, 1 mL of the Fe2SO4 solution and the ethyl salicylate solution were added respectively, and finally 1 mL of the H2O2 solution was added, mixed, reacted at 37°C for 1 h, and the absorbance was measured at 510 nm. Each group was measured in triplicate, and the relationship curve of the hydroxyl radical scavenging rate (Y) and the sample concentration (X) was drawn.

[0071] Example 6:

[0072] As shown in the following, a short peptide of Suaeda salsa was used for an external lipid-lowering experiment, and the following method was used to implement the experiment: Figures 10 to 18 The short peptide of Suaeda salsa:

[0073] 1) Effect of the short peptide of Suaeda salsa on cell survival rate:

[0074] The logarithmic growth phase HepG2 cells were inoculated in a 96-well culture plate and cultured in a cell incubator at 37°C with 5% CO2. After the cells adhered, the culture medium was removed and 180 μL of serum-free DMEM high-glucose medium was added for starvation for 12 h. The cells were divided into a blank group and an experimental group. The blank group was added with 20 μL of serum-free DMEM high-glucose medium, and the experimental group was added with different concentrations of the short peptide of Suaeda salsa (25, 50, 100, 200, 400, 800, 1600 μg / mL). After 24 h of culture, 10 μL of MTT solution with a concentration of 5 mg / mL was added, and the cells were further cultured in the cell incubator for about 3 h. Then the culture medium was removed, 150 μL of DMSO solution was added to each well, shaken uniformly, and the absorbance was measured at 595 nm. The cell survival rate was calculated.

[0075] 2) Cell grouping, modeling and drug administration:

[0076]

[0077] ​According to the results of the pre-experiment, the HepG2 cells were divided into 6 groups. The blank group was cultured in serum-free DMEM high-sugar medium (starved) for 36 h; the model group was starved for 12 h, then OA (0.2 mM) was added and cultured for 24 h; the positive control group was added with the optimal concentration of simvastatin and cultured for 12 h, then OA (0.2 mM) was added and cultured for 24 h. The high-concentration experimental group was added with 400 μg / mL of the short peptide solution of Suaeda salsa and cultured for 12 h, then OA (0.2 mM) was added and cultured for 24 h. The medium-concentration experimental group was added with 200 μg / mL of the short peptide solution of Suaeda salsa and cultured for 12 h, then OA (0.2 mM) was added and cultured for 24 h. The low-concentration experimental group was added with 100 μg / mL of the short peptide solution of Suaeda salsa and cultured for 12 h, then OA (0.2 mM) was added and cultured for 24 h.

[0078] 3) Oil red O staining:

[0079] The cultured HepG2 cells were washed with PBS twice, 2 mL of 4% neutral formaldehyde solution was added to each well, and fixed for 30 min. After the neutral formaldehyde solution was discarded, PBS was used for continuous washing twice. 1 mL of oil red O dye was added to each well, and stained for 30 min. After the oil red O dye was absorbed, PBS was used for washing again for 2-3 times. The culture dish was placed under a microscope to observe the staining effect of lipid droplets, and isopropyl alcohol was added for dissolution. The absorbance was measured at 570 nm wavelength by using an enzyme-labeled instrument.

[0080] 4) Determination of biological indicators:

[0081] The above samples were taken to measure the indicators related to lipid metabolism such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (γ-GT), superoxide dismutase (SOD), lactate dehydrogenase (LDH), triglyceride (TG), and total cholesterol (TC). The detection method was referred to the instruction manual of the kit.

[0082] As can be seen from the above, the prepared short peptide of Suaeda salsa has a molecular weight of less than 2 KDa, which is easy to be absorbed in vivo. The results of in vitro antioxidant experiment show that the short peptide of Suaeda salsa has scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and hydroxyl free radical (·OH); and the scavenging effect has a dose-dependent relationship. When the concentration of the short peptide of Suaeda salsa is 0.25, 0.4, and 0.4 mg / mL, the scavenging rates of DPPH, ABTS, and ·OH have reached 50%, which shows good antioxidant capacity.

[0083] The in vitro lipid-lowering experiment results show that in an oleic acid (OA) induced lipid peroxidation HepG2 cell model, the salt sedge short peptide prepared by the application can dose-dependently inhibit the increase of glutamic pyruvic transaminase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (gamma-GT), lactic dehydrogenase (LDH), triglyceride (TG), total cholesterol (TC) and other lipid metabolism related indexes; and the content of intracellular superoxide dismutase (SOD) is increased. Therefore, the salt sedge short peptide prepared by the application can be used for reducing the lipid accumulation of HepG2 cells caused by oleic acid (OA) stimulation. The salt sedge can be used for lipid-lowering effect research, and the short peptide component contained therein may be the main pharmacodynamic material basis.

[0084] The salt sedge short peptide sequence composition is shown in Table 1 as follows:

[0085] Table 1

[0086]

[0087]

[0088]

[0089] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0090] In the drawings of the embodiments of the present application, only the structures related to the embodiments of the present disclosure are involved, and other structures can be referred to the general design. In the case of no conflict, the same embodiment and different embodiments of the present application can be combined with each other.

[0091] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary of the principles and application of the present application. Numerous modifications and adaptions can be effected without departing from the spirit and scope of the present application, which is not limited to the exact construction and arrangement described. It is intended, therefore, to cover all modifications and adaptions that fall within the scope of the claims and their equivalents.

Claims

1. A method for preparing a short peptide of Suaeda salsa, characterized in that: The molecular weight of the Suaeda salsa short peptide is less than 2KDa, contains 48 peptide sequences, and has antioxidant activity and in vitro lipid-lowering effects.

2. The method for preparing a Suaeda salsa short peptide according to claim 1, wherein The following steps are involved: S1. Collecting Suaeda salsa leaves, washing, drying, crushing, and sieving (140 mesh) to obtain Suaeda salsa powder; S2. Add petroleum ether to the Suaeda salsa powder, mix thoroughly, let stand, filter, and discard the filtrate to obtain a Suaeda salsa filter residue; S3, weigh the Suaeda salsa filter residue obtained in step S2, add the phosphate buffer solution of 0.04mol / L with the ratio of solid-liquid ratio 1:13 (w / v), ultrasonic extraction, the extract obtained is centrifuged for 15min at 4°C, and the rotating speed is 6000r / min, then take the supernatant, add (NH4)2SO4 powder, 4°C stand, make its saturation reach 80%, after standing, centrifuged for 15min at 4°C, and the rotating speed is 6000r / min, the precipitation is dissolved with PBS buffer, put into dialysis bag and dialyzed for 24h, lyophilized, and obtain Suaeda salsa crude protein, which is stored at low temperature for standby use; S4, the crude protein powder of Suaeda salsa was prepared into 15 parts of Suaeda salsa protein aqueous solution of the same concentration, each 5 parts as a group, and a single factor experiment was carried out, setting three factors of enzyme addition amount, enzymatic hydrolysis temperature and enzymatic hydrolysis time, and 5 levels for each factor, that is, the enzyme addition amount was 4000, 6000, 8000, 10000 and 12000 U / g respectively; the enzymatic hydrolysis temperature was 45, 50, 55, 60 and 65 ° C respectively; the enzymatic hydrolysis time was 1, 2, 3, 4 and 5 h respectively, to obtain the corresponding extract; S5, centrifuging the extract obtained in step S4 to obtain a supernatant, and freeze-drying to obtain Suaeda salsa peptide powder; S6. According to the Box-Benhnken experimental design principle, the yield of short peptides from Suaeda salsa was used as the response value. The three factors of enzyme addition, hydrolysis temperature and hydrolysis time were selected to design a response surface experiment to optimize the process parameters of enzymatic hydrolysis extraction of Suaeda salsa peptides.

3. The method for preparing a Suaeda salsa short peptide according to claim 2, wherein: The enzyme addition amount is 7200 U / g, the pH value is 8.5, the enzymolysis temperature is 62° C., the enzymolysis time is 2 h, and the yield of the Suaeda salsa short peptide is 52.33%.

4. A method for determining the composition of a short peptide sequence of Suaeda salsa, characterized in that: The following steps are involved: A1. Ultrafiltration: The salsa alkali peptide solution was passed through ultrafiltration membranes with molecular weight cut-offs of 10, 5, and KDa to remove macromolecular fragments. The filtrate with a molecular weight of less than 3 KDa was concentrated and freeze-dried to obtain salsa alkali peptide powder. A2, column chromatography: The salsa salsa peptide powder obtained in step A1 was prepared into a 3 mg / mL loading solution with ultrapure water, and the treated dextran gel filler (Sephadex G-25) was loaded into a 1.6 cm × 150 cm glass chromatography column for column chromatography separation; the sample load was 3 mL, and the solution was eluted with ultrapure water at a flow rate of 20 mL / h and detected by UV detector; multiple elution peaks appeared, and the elution peaks were collected and freeze-dried respectively. The antioxidant activity and in vitro lipid-lowering effect of each component were determined, and the component with the highest activity was obtained and stored at low temperature for later use; A3. Sequence Identification: The sample obtained in step A2 was separated by RP-HPLC and then analyzed by mass spectrometry using a Q-Exactive mass spectrometer. Analysis conditions: positive ion detection; mass-to-charge ratio (m / z) acquisition method for peptides and peptide fragments: 10 fragment spectra were collected after each full scan; analysis time was 60 min.

5. The method for determining the short peptide sequence composition of Suaeda salsa according to claim 4, wherein: The short peptide from Suaeda salsa has scavenging effects on 1,1-diphenyl-2-trinitrophenylhydrazine, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and hydroxyl radicals. Used to reduce lipid accumulation in HepG2 cells caused by oleic acid stimulation; Reduces intracellular alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transpeptidase, lactate dehydrogenase, triglycerides, total cholesterol and lipid metabolism-related indicators; increases the content of superoxide dismutase.

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