Antarctic krill active peptide, preparation method and application thereof

Abstract

The present application relates to the field of polypeptides, in particular to a euphausia superba active peptide and a preparation method and application thereof.The sequence of the euphausia superba active peptide comprises at least one of LFP, FDL and FSL.The euphausia superba active peptide has good anti-inflammatory effect and can be used for preparing a euphausia superba selenium chelate peptide.The prepared euphausia superba selenium chelate peptide has good effect in treating ulcerative colitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of polypeptides, in particular to a Euphausia superba active peptide and a preparation method and application thereof. BACKGROUND

[0002] Euphausia superba as a potential sustainable marine resource, because its protein content is more than 60%, it is concerned. Enzymatic hydrolysis of active peptides (AKPs) has been confirmed to have antioxidant, anti-inflammatory (inhibition of TNF-α / IL-1β) and immune regulation and other multiple biological activities. Based on the "natural ingredients synergistic effect" strategy, this study proposes an innovative idea: AKPs as molecular carriers, through the carboxyl / amino and other active groups chelate selenium ions, construct Euphausia superba selenium chelate peptides (Se-AKPs). Peptide-selenium chelate can be actively absorbed by intestinal oligopeptide transporter (PEPT1), significantly improve the stability and bioavailability of selenium. The annual capture of Euphausia superba is more than 500,000 tons, and the development of high value selenium peptide products meets the "blue granary" national strategy.

[0003] Selenium (Se) as an essential trace element, through the integration into the active center of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), remove reactive oxygen species (ROS), NF-κB signaling pathway, thus play a core anti-inflammatory and antioxidant effect. Although selenium plays an important role in health, but the traditional selenium supplements still have significant defects. Inorganic selenium is absorbed by passive diffusion, bioavailability is only 30-50%, and more than physiological dose will produce liver toxicity and genetic toxicity. Although active transport absorption (utilization rate > 80%) can be used for organic selenium, but the synthesis cost is high and the anti-inflammatory activity is limited. The biological utilization barrier of selenium is essentially due to the instability of its chemical form and insufficient targeting delivery efficiency. Therefore, it is urgent to design new selenium carriers to enhance the biological safety and anti-inflammatory efficacy.

[0004] Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a global disease characterized by chronic inflammation of the intestinal tract. Epidemiological data show that the global incidence of IBD is increasing, and the number of UC patients in China has increased significantly in recent years, becoming a public health challenge. Its pathological mechanism involves the interaction of multiple factors such as genetic susceptibility, intestinal mucosal barrier defects, and immune regulation abnormalities. Patients show repeated diarrhea, hematochezia, and abdominal pain, which seriously affect the quality of life. Current clinical treatment has certain limitations, 5-aminosalicylic acid (5-ASA) is used for mild to moderate patients, but 30% of patients are ineffective and accompanied by side effects such as headache and skin rash; glucocorticoid-induced remission is required for moderate to severe patients, but long-term use can easily cause infection and osteoporosis; biological agents (such as anti-TNF-α monoclonal antibodies) can promote mucosal healing, but there are problems of high price and secondary failure; the hepatotoxicity of immunosuppressive agents (such as azathioprine) and the risk of pouchitis (50% incidence) of surgical treatment further highlight the limitations of treatment. Therefore, it is urgent to develop safe and efficient new anti-inflammatory strategies.

[0005] In this study, the effect of Antarctic krill selenium-chelating peptide on improving inflammation was explored. First, Antarctic krill selenium-chelating peptide was prepared, then the selenium-chelating rate was determined, and finally the anti-inflammatory effect was analyzed by using a cell model and an animal model. This study first systematically explores the structure-activity relationship of Se-AKPs and their potential for treating UC, providing a dual solution for IBD drug development and selenium nutrition innovation. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application discloses a kind of Antarctic krill active peptide and its preparation method and application, content is as follows specifically:

[0007] In a first aspect of the application, a kind of Antarctic krill active peptide is provided, and the sequence of the Antarctic krill active peptide includes at least one of LFP, FDL and FSL.

[0008] In a second aspect of the application, a method for preparing the Antarctic krill active peptide is provided, and the method includes at least one of solid-phase polypeptide synthesis, liquid-phase polypeptide synthesis and enzymatic hydrolysis.

[0009] Further, the protease in the enzymatic hydrolysis includes at least one of acid protease, neutral protease, papain, alkaline protease, trypsin and pepsin.

[0010] In a specific embodiment of the application, the protease is alkaline protease.

[0011] In a third aspect of the application, the Antarctic krill active peptide is applied in any of the following aspects:

[0012] A1. Application in preparing a product for inhibiting NO release;

[0013] A2. Use in the preparation of an anti-inflammatory product;

[0014] A3. Use in the preparation of Euphausia superba metal-chelating peptide.

[0015] Further, the Euphausia superba metal-chelating peptide in A3 includes at least one of Euphausia superba ferrous-chelating peptide, Euphausia superba zinc-chelating peptide, Euphausia superba selenium-chelating peptide, and Euphausia superba calcium-chelating peptide.

[0016] In a specific embodiment of the present application, the Euphausia superba metal-chelating peptide is Euphausia superba selenium-chelating peptide.

[0017] In a fourth aspect of the present application, a Euphausia superba selenium-chelating peptide is provided, which includes at least Euphausia superba selenium-chelating peptide prepared by using the Euphausia superba active peptide.

[0018] In a fifth aspect of the present application, a method for preparing the Euphausia superba selenium-chelating peptide is provided, which includes at least the following steps:

[0019] S1. Adding at least one of acid protease, neutral protease, papain, alkaline protease, trypsin, and pepsin to a Euphausia superba protein solution to obtain a Euphausia superba proteolysis solution;

[0020] S2. Adding Na2SeO3 to the Euphausia superba proteolysis solution at a ratio (v / v) of 2:1 to 1:3 to obtain a Euphausia superba selenium-chelating peptide.

[0021] Further, the activity of the protease is between 50 k and 2000 k U / g.

[0022] Further, the method further includes the step of homogenizing the Euphausia superba shrimp meat and extracting Euphausia superba protein by centrifugation.

[0023] Further, the specific operation of the step of homogenizing the Euphausia superba shrimp meat and extracting Euphausia superba protein by centrifugation is as follows:

[0024] Weigh the Euphausia superba shrimp meat, and homogenize it with deionized water at a liquid-to-material ratio of 2.5 to 3.5 mL / g. Then, adjust the pH to 11 to 12, and then centrifuge to obtain supernatant. Optionally, the extraction step can be repeated 3 to 5 times. Finally, after combining the supernatants, adjust the pH of the collected supernatant to 4.5, centrifuge again, collect the precipitate, and freeze-dry and store.

[0025] In a sixth aspect of the present application, the Euphausia superba selenium-chelating peptide is used in any of the following aspects:

[0026] B1. Use in the preparation of a product for inhibiting NO release;

[0027] B2. Use in the preparation of an anti-inflammatory product.

[0028] Further, the Euphausia Superba selenium-chelated peptides are used for preparing a medicine for treating ulcerative colitis.

[0029] The beneficial effects of the present application include but are not limited to:

[0030] The Euphausia Superba active peptides LFP, FDL and FSL disclosed in the present application can inhibit the NO release amount of RAW264.7 cells. The composite peptide segments LFP+FDL, LFP+FSL and FDL+FSL can significantly inhibit the release of NO, and the NO release amount is obviously reduced compared with that of a single peptide segment.

[0031] In the present study, Euphausia Superba selenium-chelated peptides (Se-AKPs) were successfully prepared, and their significant anti-inflammatory activity was confirmed. In vitro experiments showed that Se-AKPs could effectively inhibit the release of NO in RAW264.7 cells induced by LPS. Animal experiments further demonstrated that Se-AKPs could significantly alleviate DSS-induced ulcerative colitis in mice, improve disease activity index, body weight loss and colon shortening, and regulate serum inflammatory factor levels. This study provides a theoretical basis and application potential for the development of new anti-inflammatory strategies and IBD treatment based on selenium-chelated peptides. BRIEF DESCRIPTION OF DRAWINGS

[0032] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0033] Figure 1 is a schematic diagram of the determination results of the hydrolysis degree of Euphausia Superba protein by different proteases in the embodiments of the present application.

[0034] Figure 2 is a schematic diagram of the chelation rate of Euphausia Superba polypeptides and selenium by different proteases in the embodiments of the present application.

[0035] Figure 3 is the ultraviolet absorption spectrum of AKPs and Se-AKPs in the embodiments of the present application in the wavelength range of 200 ~ 600 nm.

[0036] Figure 4 is the fluorescence spectrum of AKPs and Se-AKPs in the embodiments of the present application in the wavelength range of 300 ~ 500 nm.

[0037] Figure 5is the CD spectrum (Figure A) and the secondary structure distribution diagram (Figure B) of AKPs and Se-AKPs in the embodiment of the present application.

[0038] Figure 6 is the particle size distribution curve (Figure A) and the Zate potential structure diagram (Figure B) of AKPs and Se-AKPs in the embodiment of the present application.

[0039] Figure 7 is the SEM electron microscope diagram of AKPs (Figure A) and Se-AKPs (Figure B) in the embodiment of the present application.

[0040] Figure 8 is the result diagram of the influence of different concentrations of LPS on cell viability in the embodiment of the present application.

[0041] Figure 9 is the result diagram of the influence of different concentrations of LPS on NO release in the embodiment of the present application.

[0042] Figure 10 is the influence diagram of different concentrations of AKPs and Se-AKPs on the cell viability of mouse macrophage RAW264.7 cells in the embodiment of the present application, Figure A is the influence diagram of AKPs on the cell viability of mouse macrophage RAW264.7 cells, and Figure B is the influence diagram of Se-AKPs on the cell viability of mouse macrophage RAW264.7 cells.

[0043] Figure 11 is the influence diagram of different concentrations of AKPs and Se-AKPs on the LPS-induced RAW264.7 cell toxicity in the embodiment of the present application, Figure A is the influence diagram of AKPs on the LPS-induced RAW264.7 cell viability, and Figure B is the influence diagram of Se-AKPs on the LPS-induced RAW264.7 cell viability.

[0044] Figure 12 is the influence diagram of different concentrations of AKPs (Figure A) and Se-AKPs (Figure B) on the NO release of LPS-induced RAW264.7.

[0045] Figure 13 is the change diagram of the disease activity index (DAI) of each group after DSS induction in the embodiment of the present application.

[0046] Figure 14 is the result diagram of the daily body weight change of mice in each experimental group in the embodiment of the present application.

[0047] Figure 15is the photo of the stool consistency and fecal occult blood of the mice in the Control, DSS, AKPs, Se-AKPs, DSS+drug groups in the embodiment of the present application.

[0048] Figure 16 is the diagram of the colon length of the mice in the Control, DSS, AKPs, Se-AKPs, DSS+drug groups in the embodiment of the present application, Fig. A is the photo of the colon of the mice, and Fig. B is the statistical diagram.

[0049] Figure 17 is the diagram of the levels of IL-6 (Fig. A), IL-10 (Fig. B), IL-1β (Fig. C), TNF-α (Fig. D), and CPR (Fig. E) in the serum in the embodiment of the present application.

[0050] Figure 18 is the staining diagram of the effect of Se-AKPs on the colon pathology of the mice in the embodiment of the present application.

[0051] Figure 19 is the diagram of the anti-inflammatory activity of the Antarctic krill selenium-chelated peptides with different molecular weights in the embodiment of the present application.

[0052] Figure 20 is the diagram of the secondary mass spectrometry of the Antarctic krill active peptides FDL (Fig. A), FSL (Fig. B), and LFP (Fig. C) in the embodiment of the present application.

[0053] Figure 21 is the diagram of the effect of the Antarctic krill active peptides LFP, FDL, and FSL on the activity of RAW264.7 cells in the embodiment of the present application, wherein Fig. A is the diagram of the effect of the Antarctic krill active peptides LFP, FDL, and FSL on the activity of RAW264.7 cells, and Fig. B is the diagram of the effect of the Antarctic krill active peptides LFP, FDL, and FSL on the activity of RAW264.7 cells.

[0054] Figure 22 is the diagram of the effect of the Antarctic krill active peptides LFP, FDL, and FSL on the NO release amount of RAW264.7 cells in the embodiment of the present application, wherein Fig. A is the diagram of the effect of the Antarctic krill active peptides LFP, FDL, and FSL on the NO release amount of RAW264.7 cells, and Fig. B is the diagram of the effect of the Antarctic krill active peptides LFP, FDL, and FSL on the NO release amount of RAW264.7 cells. DETAILED DESCRIPTION

[0055] The present application will be described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present application are all purchased through commercial channels.

[0056] Preparation of Antarctic krill active peptides

[0057] The Antarctic krill meat was weighed and homogenized in a beaker with deionized water (4 °C) at a liquid-to-material ratio of 3.17 mL / g. Subsequently, the pH was adjusted to 11.38 with 2 mol / L sodium hydroxide, and after standing for 0.5 h, centrifugation was performed at 10,000 g for 10 min at 4 °C to obtain the supernatant. The entire extraction process was repeated three times. Finally, the collected supernatant was adjusted to pH 4.5 with 2 mol / L phosphoric acid, and after standing for 1.0 h, centrifugation was performed at 10,000 g for 10 min at 4 °C to collect the precipitate, which was stored by freeze-drying at -20 °C. The protein content was determined, and the protein recovery rate was calculated.

[0058] The extracted Antarctic krill protein was dissolved in deionized water at a concentration of 2% (w / v), and six different proteases at a concentration of 2% (w / v) were added to the solution. The entire enzymatic reaction was performed in a shaking water bath incubator, and the hydrolysis time was 4 h for each protease to obtain Antarctic krill active peptides (AKPs). Subsequently, the hydrolysate was placed in a boiling water bath for 15 min and quickly cooled to 4.0 °C with ice water. Centrifugation was performed at 12,000 g for 15 min. The supernatant was collected, and the degree of hydrolysis was determined before freeze-drying. The optimal reaction conditions for the six proteases are shown in Table 1.

[0059]

[0060]

[0061] The degree of hydrolysis of Antarctic krill protein hydrolyzed by the six proteases under the optimal reaction conditions was calculated as follows:

[0062] After the enzymatic reaction, the supernatant was centrifuged and immediately subjected to the determination of the degree of hydrolysis. The degree of hydrolysis of Antarctic krill protein was determined using the o-phthaldialdehyde (OPA) method. Specifically, 80 mg of OPA was dissolved in 2 mL of absolute ethanol, 200 μL of β-mercaptoethanol, 5 mL of 10% SDS (w / v), and 92.8 mL of 0.1 mol / L sodium tetraborate to prepare 100 mL of OPA reagent solution. Forty μL of Antarctic krill hydrolysate was mixed with 4 mL of OPA reagent at room temperature for 2 min, and the absorbance at 340 nm was measured. Antarctic krill protein was placed in 6 mol / L HCl and reacted at 115 °C for 24 h. The number of free amino groups was determined using a standard curve of serine as the number of free amino acids produced by complete hydrolysis of Antarctic krill protein.

[0063] The degree of hydrolysis of Antarctic krill protein was calculated according to the following formula:

[0064] Hydrolysis degree (%) = [(NH2) t -(NH2) t0 )] / (NH2) T

[0065] In the formula: (NH2) t represents the amount of free amino acid released at t; (NH2) t0 represents the amount of free amino acid without enzyme hydrolysis; (NH2) T represents the amount of free amino acid after complete hydrolysis.

[0066] The results are shown in Table 1. Figure 1 As shown in Table 1, compared with other four proteases, alkaline protease and trypsin have higher hydrolysis degree of Antarctic krill protein, which are 37.77% and 33.75% respectively. This may be because under the condition of sufficient substrate, there are more enzyme cleavage sites for these two enzymes, and the protein is more easily hydrolyzed into small molecular peptides. Compared with other five proteases, alkaline protease is the most optimal.

[0067] Example 2 Preparation of Antarctic krill selenium-chelated peptides

[0068] Antarctic krill selenium-chelated peptides (Se-AKPs) were prepared by dropping the Antarctic krill active peptides (AKPs) obtained in Example 1 into a 0.1 mol / L Na2SeO3 solution. In the chelating system, 0.1 mol / L sodium selenite solution and 3% (W / V) Antarctic krill protein hydrolysate were fully mixed at a volume ratio of 1:2 to prepare Antarctic krill selenium-chelated peptides, and the pH was adjusted to 9.0. Then the reaction was carried out in a water bath at 80 ℃ for 1 h, cooled to room temperature, and after centrifugation, the supernatant was taken and 5 times the volume of 95% ethanol solution was added, mixed uniformly and then left to precipitate for 12 h, and after centrifugation, the precipitate was collected. Finally, the precipitate was washed with a small amount of anhydrous ethanol to remove the unbound selenium, and the precipitate was freeze-dried to obtain Antarctic krill selenium-chelated peptides, which were ready for use.

[0069] Test Example 1 Determination of chelation rate of Antarctic krill selenium-chelated peptides

[0070] This method uses atomic fluorescence spectrometer to accurately determine the selenium chelation rate by hydride generation-atomic fluorescence spectrometry (HG-AFS). First, the chelate is taken in equal amount, digested by microwave, and then diluted with 5% HCl to obtain total selenium solution; 6 mol / L HCl is added to the digestion solution and reduced at 95 ℃ water bath for 30 min, and Se 6+ is converted to Se 4+To optimize hydrogenation efficiency, HG-AFS detection was subsequently employed (excitation wavelength 196 nm, atomizer temperature 200 °C, argon carrier gas flow rate 400 mL / min). Using 0.8% KBH4 (containing 0.5% NaOH) as the reducing agent and 5% HCl as the carrier liquid, the hydrogenation efficiency was determined using a selenium standard curve (0–20 μg / L, R0). 2 >0.995) The quantitative formulas for free selenium and total selenium concentration are as follows:

[0071] Chelation rate (%) = (1 - Free selenium concentration / Total selenium concentration) × 100%

[0072] The experiment included spiked recovery (85–115%) and parallel determination (RSD < 5%), and the accuracy was verified using NIST SRM 1549 standard material. The method detection limit was 0.1 μg / L, which can accurately determine the binding efficiency of active selenium in the selenopeptide complex.

[0073] The results are as follows Figure 2 As shown, the chelation rate of peptides (neutral protease, alkaline protease, trypsin, pepsin, acidic protease, and papain) after enzymatic hydrolysis with six enzymes was determined by chelating them with selenium under optimal conditions. Figure 2 It can be seen that the chelating ability of alkaline protease is higher than that under other conditions, with a chelation rate of 41.8%. Therefore, alkaline protease was chosen for enzymatic hydrolysis of Antarctic krill protein for chelation. Alkaline protease exhibits the highest activity under alkaline conditions. Under this pH environment, Antarctic krill protein may be more easily hydrolyzed into a large number of small peptides with specific sequences and structures (such as those rich in -His, -Lys, and -Glu amino acids). The functional groups on these peptides are more likely to bind with selenium ions (Se). 4+ The peptides combine to form stable chelates. Therefore, Antarctic krill selenium chelate peptides prepared from Antarctic krill bioactive peptides hydrolyzed by alkaline protease were selected for subsequent experiments.

[0074] Test Example 2: Physicochemical Properties of Selenium Chelated Peptides from Antarctic Krill

[0075] (1) Ultraviolet-visible absorption spectrum

[0076] The lyophilized AKPs and Se-AKPs powders were dissolved in deionized water at a concentration of 0.1 mg / mL. The UV-Vis spectra of AKPs and Se-AKPs in the wavelength range of 200–600 nm were recorded using a UV-Vis spectrophotometer (Perkin Elmer, Salem, MA).

[0077] from Figure 3As can be seen from Figure 1, the absorption peak of AKPs is the strongest at 270 nm, while the absorption peak of Se-AKPs is red-shifted to 280 nm and the absorption peak intensity is increased. The shift of the peak and the change of the peak intensity can be used as a direct indicator of the successful chelation of selenium with Antarctic krill protein, and the chelation site is most likely to act on sulfur-containing groups such as cysteine.

[0078] (2) Fluorescence spectrum

[0079] The freeze-dried AKPs and Se-AKPs were dissolved in deionized water with a concentration of 0.1 mg / mL. The fluorescence intensity of AKPs and Se-AKPs at 290-500 nm was measured under an excitation wavelength of 288 nm.

[0080] The fluorescence spectra of AKPs and Se-AKPs in the wavelength range of 290-500 nm are shown in Figure 2. Figure 4 The results show that the combination of selenium with AKPs leads to a significant decrease in fluorescence intensity, which is due to the folding and aggregation of selenium with some color-forming amino acids (such as tyrosine and tryptophan) in the peptide during chelation. Moreover, the addition of mineral ions can also lead to fluorescence quenching of the sample.

[0081] (3) Circular dichroism (CD)

[0082] AKPs and Se-AKPs at 1 mg / mL were placed in a quartz test tube with an optical path of 0.1 cm, and a Jasco J-1500 circular dichroism spectrometer was used to repeatedly scan 3 times at a wavelength of 190-260 nm at a speed of 1 nm / s. The scanning temperature was (25±0.2) °C.

[0083] The results are shown in Figure 3. Figure 5 As can be seen from Figure 3, AKPs have a strong negative peak at 210 nm, indicating that AKPs have an ordered β-sheet structure. The absorbance rises rapidly after the 210 nm valley, indicating the presence of a small amount of α-helix or superimposed random coil. The absorbance of Se-AKPs at 210 nm is close to 0, and the overall curve is flat, indicating that selenium chelation significantly reduces the β-sheet structure and changes to disorder or random coil. This indicates that selenium chelation induces a change from an ordered secondary structure to a random coil-dominated flexible conformation by destroying the AKPs. This structural rearrangement can enhance the dynamic nature of the molecule and the exposure of antioxidant groups, thereby improving its biological activity.

[0084] (4) Particle size / Zeta potential analysis

[0085] AKPs and Se-AKPs were dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL, then added to the U-shaped sample cell, stabilized at 25 °C for 5 s, and the particle size and zeta potential of AKPs and Se-AKPs were determined using a Zetasizer Nano ZS90 nanoparticle size potential analyzer.

[0086] The results, as shown in Figure 6 , the average particle size of AKPs and Se-AKPs was 608.23±2.65 nm and 660.09.51±1.18 nm, respectively. The particle size of the Se-AKPs group was significantly larger than that of the AKPs group. This may be due to the growth of the peptide chain caused by the chelation of protein at the end of the peptide chain with Se or metal ion elements, resulting in structural folding and aggregation reactions, thereby leading to an increase in the particle size of Se-AKPs. The PDI of Se-AKPs (0.65±0.09) was lower than that of AKPs (0.82±0.12), indicating that Se-AKPs were more evenly dispersed.

[0087] The zeta potential of AKPs and Se-AKPs was 6.93 mV and -35 mV, respectively. The zeta potential value of Se-AKPs was significantly reduced, which may be due to the electron transfer during the reaction of polypeptides with Se 2+ . After Se 2+ was chelated by AKPs, the more electronegative group was inside, and due to the Se 2+ repulsion, the positively charged group was on the outside, resulting in a large number of negative charges around Se-AKPs, with a negative zeta potential. This indicates that Se-AKPs do not exist in the form of a double electric layer, but in the form of a molecular structure. Therefore, Se 2+ may have been surrounded by the functional binding sites of AKPs, including carbonyl, amino, and carboxyl groups, showing that it is a neutral molecule rather than a coordination bond in the form of inorganic selenium to increase its stability in the gastrointestinal tract.

[0088] (5) Scanning electron microscopy (SEM)

[0089] A suitable amount of completely dried AKPs and Se-AKPs chelate powder was adhered to the sample stage with conductive glue, lightly blown with an ear bulb, and then coated with a gold film using ion sputtering, and observed and photographed under a field emission scanning electron microscope.

[0090] The results, as shown in Figure 7As shown, selenium chelation disrupts the hydrogen bond network of Antarctic krill bioactive peptides and introduces molecular cross-linking, leading to a transformation of their microstructure from an interconnected, irregular granular structure to a porous, amorphous, and highly irregular sponge-like structure. This morphological change originates from selenium atoms disrupting the peptide chain's hydrogen bond network, inducing molecular cross-linking and disordering, exposing hydrophobic groups, and enhancing specific surface area and accessibility of active sites. This indicates that selenium chelation reshapes the structure-activity relationship of peptides.

[0091] Test Example 3: In vitro anti-inflammatory experiment of Antarctic krill selenium chelate peptides

[0092] (1) Cell culture

[0093] Mouse macrophages RAW264.7 were cultured in DMEM high-glucose complete medium (supplemented with 10% fetal bovine serum and 0.1 mg / mL streptomycin and 0.1 mg / mL penicillin). Cells were cultured in a constant temperature (37°C) in an incubator with 95% humidity and 5% carbon dioxide until the logarithmic growth phase was reached for experimental use.

[0094] (2) Screening of LPS concentration

[0095] Cells in the logarithmic phase were divided into groups of 5 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL per well in 96-well plates. The plates were incubated overnight. After cell attachment (usually 12–24 h), the old culture medium was discarded, and then 100 μL of LPS at different concentrations (0, 0.1, 0.5, 1, 1.5 μg / mL) was added to each well for 24 h of stimulation. Cell viability and NO content were then measured.

[0096] Cell viability assay results as follows Figure 8 As shown, when the concentrations of LPS were 0.1 μg / mL, 0.5 μg / mL, and 1 μg / mL, cell viability increased to varying degrees, demonstrating that within this range, LPS had no toxic effect on RAW264.7 macrophages. However, when the LPS concentration was 1.5 μg / mL, cell viability was 72.9%, indicating that LPS at this concentration had some toxic effect on RAW264.7 macrophages.

[0097] The NO content results are as follows: Figure 9 As shown, RAW264.7 macrophages were stimulated with different concentrations of LPS for 24 h. It was found that NO release was significantly increased when the LPS concentration was 1-1.5 μg / mL. Considering all factors, 1 μg / mL of LPS was selected as the effective concentration for establishing the cellular inflammation model.

[0098] (3) Measurement of cell viability

[0099] Cell viability was determined using CCK-8. Trypsin digestion was used to detach the cells in the logarithmic phase, and the cells were diluted to a density of 5 x 10 4 450

[0100] Cell viability (%) = [A (drug) - A (blank)] / [A (0 drug) - A (blank)] x 100%

[0101] A (drug): absorbance of the well with cells, CCK-8 solution, and drug solution; A (blank): absorbance of the well with medium and CCK-8 solution without cells; A (0 drug): absorbance of the well with cells, CCK-8 solution, and no drug solution.

[0102] The results are shown in Figure 10 AKPs had no toxic side effects on RAW264.7 macrophages at concentrations of 0.5-5 mg / mL. Se-AKPs had no toxicity to RAW264.7 macrophages at a concentration of 0.1 mg / mL and had a certain proliferation effect on cell growth. In addition, other Se-AKPs had significant toxic effects on RAW264.7 macrophages (p < 0.05) as the concentration of the enzymatic hydrolysate increased in the range of 0.3-5 mg / mL. Therefore, the concentration of Se-AKPs was selected as 0.1 mg / mL in the subsequent experiments.

[0103] To determine the safe concentration range of Se-AKPs on RAW264.7 cells induced by 1 μg / mL of LPS, the results are shown in Figure 11 ​​As shown, LPS had no effect on the survival rate of mouse macrophage RAW264.7 compared with the control group (0 mg / mL). In addition, different AKPs had no effect on LPS-induced RAW264.7 macrophages in the concentration range of 0.1-5 mg / mL, indicating that the concentration range of 5 mg / mL was a safe and effective range. Se-AKPs were non-toxic to LPS-induced RAW264.7 macrophages at 0.1 mg / mL and had a certain proliferation effect on cell growth. In summary, 0.1 mg / mL was selected as the safe concentration for use.

[0104] (4) Determination of nitric oxide (NO) content

[0105] The content of NO was detected by the Griess method. Cells in the logarithmic phase were inoculated in a 96-well plate at a density of 5x10 4 cells / well in 100 μL. The culture plate was placed in an incubator for overnight culture, and after the cells adhered (usually 12-24 h), the old liquid in the culture plate was discarded, and then 100 μL of LPS and phycocyanobilin proteolysis liquid of different concentrations was added to each well for incubation for a specified time. After the culture ended, the cell culture supernatant was collected, and the NO content was determined using a NO detection kit. Briefly, first restore Griess Reagent I and II to room temperature. According to the sample type, select the corresponding diluent to prepare the NO standard gradient. Add 50 μL of standard and treated sample to each well of a 96-well plate. Then add 50 μL of Griess Reagent I and II, mix well, and measure the absorbance at 540 nm. Using sodium nitrite (NaNO2) standard, a standard curve (0-100 μmol / L) was prepared, and the content of NO was calculated according to the standard curve.

[0106] The results are shown in Figure 12 Compared with the blank group (0 mg / mL), the release of NO from RAW264.7 macrophages stimulated by LPS was significantly increased (p<0.05), indicating that the inflammatory model induced by LPS was successfully established. AKPs and Se-AKPs both reduced the release of NO and enhanced the anti-inflammatory effect with increasing concentration. Se-AKPs had very low NO release at 0.3-5 mg / mL, which may be due to the significant toxic effect of Se-AKPs on RAW264.7 macrophages at 0.3-5 mg / mL. In summary, 0.1 mg / mL was selected as the safe concentration.

[0107] Test 4 Antarctic krill selenium-chelated peptide animal experiment

[0108] (1) Establishment of animal model

[0109] Mice were housed in a SPF environment with independent ventilation system, 12-hour light-dark cycle, relative humidity of 55±5%, food and water freely available, and acclimated for one week before being housed in cages. The mouse cages were disinfected and the bedding was changed three times a week. The test animals were divided into 5 groups, 6 mice in each group, and different drugs were treated by gavage at a fixed time every day. The experimental grouping is as follows:

[0110] Control group (Control group): free drinking of sterile distilled water for 7 days, and gavage of sterile distilled water at the same time;

[0111] Model group (DSS group): free drinking of 3% DSS solution to construct ulcerative colitis model for 7 days, and gavage of sterile distilled water at the same time;

[0112] DSS+AKPs group: free drinking of 3% DSS solution to construct ulcerative colitis model for 7 days, and gavage of AKPs at the same time;

[0113] DSS+Se-AKPs group: free drinking of 3% DSS solution to construct ulcerative colitis model for 7 days, and gavage of Se-AKPs at the same time;

[0114] DSS+drug group (Positive control): free drinking of 3% DSS solution to construct ulcerative colitis model for 7 days, and gavage of mesalazine enteric-coated tablets at the same time.

[0115] The body weight, fecal bleeding, fecal consistency, disease activity index, etc. of the mice were recorded to evaluate the severity of colitis. Finally, after gavage for seven days, the mice were fasted for one day, and all the mice were euthanized on the eighth day. Serum, colon tissue, cecum, etc. were collected for subsequent analysis.

[0116] (2) Disease activity index

[0117] From the 0th day, the diet, activity, anal condition and other conditions of the mice were regularly monitored and recorded, the body weight of the mice was recorded, the feces of the mice were observed, and the disease index (Disease activity index, DAI) of the mice was scored according to the body weight, fecal blood and fecal morphology.

[0118] Table 2 DAI score table

[0119]

[0120] (3) Mouse dissection and tissue collection

[0121] After the experiment, the mice were sacrificed, dissected and tissue samples were taken. The serum was collected by centrifugation at 3000 rpm for 15 min and stored at -80°C for later use. The colon and rectum were stripped, the colon tissue of the mice was observed, the intestinal contents were observed, and the total length was measured. The colon tissue was gently washed with a normal saline buffer and dried with filter paper, and the colon tissue was weighed and the data was recorded. Then the colon tissue was cut into several sections. One part was fixed with 4% paraformaldehyde, and the other parts were cut into small pieces and frozen in liquid nitrogen, and stored at -80°C for later use.

[0122] (4) Evaluation of serum cytokines in mice

[0123] The levels of inflammatory factors interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and C-reactive protein (CRP) in serum were detected using corresponding enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.

[0124] (5) H&E staining

[0125] The colon tissue was dehydrated using different concentrations of ethanol solution gradient, transparentized using ethanol xylene, embedded after paraffin immersion, sectioned (3 μm), and baked. After staining with hematoxylin staining solution and then staining with 0.5% eosin staining solution, the sections were mounted with neutral resin. The structure was observed and photographed using a microscope, and the histological score of colitis was calculated, and the crypt depth was calculated.

[0126] The results are as follows:

[0127] (1) Disease activity index of mice

[0128] The results are shown in Figure 13 The mental state, rectal bleeding and stool consistency of the mice were observed. During the experiment, the mental state of the mice in the blank group was good, active, and the stool was normal granular. Compared with the blank group, the mice in the DSS group began to show mental fatigue, mild diarrhea, and soft stool on the fourth day after DSS treatment. After the eighth day, the mice in the DSS group showed mental fatigue, body curling, and different degrees of watery stool, anal bleeding and other symptoms. Compared with the DSS group, the drug group, AKPs group and Se-AKPs group can alleviate the above symptoms induced by DSS. Therefore, the disease activity index (DAI) of mice was comprehensively evaluated, and the results showed that the AKPs group and Se-AKPs group can alleviate the decrease of DAI score caused by DSS.

[0129] (2) Daily body weight change of mice

[0130] Results as shown in Figure 14 Figure 14, mice were induced with dextran sulfate sodium (DSS) to establish ulcerative colitis (UC) model. After 7 days of adaptation, all C57BL / 6J mice were randomly given 3% DSS for 7 days to establish UC model. The effects of drug group, AKPs group and Se-AKPs group on UC mice were observed. During the experiment, the body weight change of mice was monitored. As shown in Figure 14, the body weight of mice in normal blank group showed a slow growth trend during the experiment. Compared with the blank group, the body weight of mice in DSS model group began to decrease on the fourth day of DSS modeling. With the prolongation of the experiment, the body weight of mice in DSS group, drug group, AKPs group and Se-AKPs group showed a overall downward trend, while the drug group, AKPs group and Se-AKPs group could slow down the DSS-induced weight loss.

[0131] (3) Fecal consistency and fecal occult blood of mice

[0132] According to Figure 15 the fecal samples and the appearance of the anal region of the five groups of mice, the severity of colitis and the intervention effect of each group can be clearly reflected:

[0133] Blank group: feces showed regular black granular shape, and the anus was clean without redness, indicating a healthy intestinal tract without inflammation or bleeding.

[0134] DSS group (model group): feces were rare and loose, mixed with obvious fresh red blood, and the anus was red and swollen, which was consistent with the typical characteristics of ulcerative colitis, indicating that the modeling was successful.

[0135] Drug group (Positive Control Mesalazine intervention): feces showed blocky shape with a small amount of red material, and the anus was not significantly red and swollen, indicating that the drug partially inhibited bleeding, but there was still mild inflammation remaining.

[0136] AKPs group: a small amount of red was visible in the feces, and the anus was red and swollen, indicating that phosphorus shrimp peptide had certain anti-inflammatory and hemostatic effects, but the effect was weaker than that of the drug group.

[0137] Se-AKPs group: a small amount of red was visible in the feces, but the degree of anal redness was lighter than that of the AKPs group, which may be related to the temporary bleeding during the mucosal repair process promoted by selenium.

[0138] (4) Colon length of mice

[0139] Results as shown in Figure 16As shown, colon shortening is one of the typical symptoms of ulcerative colitis. Compared with the control group, DSS caused colon length shortening, blood in the colon and cecum, and thickening of the intestinal contents. AKPs, Se-AKPs, and drug intervention significantly alleviated the shortening of the colon length and protected against DSS-induced colon shortening.

[0140] (5) Evaluation of serum cytokines

[0141] Results, as shown in Figure 17 To further evaluate the effects of Se-AKPs on systemic and intestinal inflammation in colitis mice, we measured the levels of pro-inflammatory cytokines TNF-a, IL-6, IL-1b, CPR and anti-inflammatory cytokine IL-10 in the serum of mice using ELISA. Compared with the Control group, the levels of pro-inflammatory cytokines TNF-a, IL-6, and IL-1b, CPR were significantly increased (p<0.05), while the anti-inflammatory cytokine (IL-10) was significantly decreased in the DSS group, indicating that DSS induced inflammation in mice. After treatment with AKPs and Se-AKPs, the levels of pro-inflammatory cytokines TNF-a, IL-6, IL-1b, CPR in the serum of colitis mice were decreased, and the level of anti-inflammatory cytokine IL-10 was increased, indicating that Se-AKPs, AKPs and drugs can alleviate the inflammatory state of colitis mice. And Se-AKPs is the most effective.

[0142] (6) Evaluation of serum cytokines

[0143] Results, as shown in Figure 18As shown, DSS successfully induced colitis by observing H&E staining sections. The blank group was normal colonic mucosa structure, the model group had a large number of inflammatory cell infiltration in the lamina propria, the crypt structure was severely damaged, and the goblet cells were significantly reduced or completely lost, which proved that the 3% DSS modeling was successful. Compared with the DSS model group, the total number of inflammatory cells in the lamina propria of the AKPs group should have decreased significantly, the crypt abscesses may decrease or disappear, but still more than the blank group, and the number of goblet cells may increase to a certain extent compared with the model group, but may still not reach the normal level of the blank group, indicating that AKPs have certain anti-inflammatory and protective effects on intestinal mucosa, and can slightly alleviate DSS-induced colitis. The Se-AKPs group was better than the AKPs group in terms of inflammatory cell infiltration, crypt arrangement was neat, structure was complete, goblet cell number was rich, and morphology was close to normal, repair effect was better than AKPs group, and selenium may enhance the biological activity of the peptide. The drug group as a first-line drug for clinical treatment of UC can effectively inhibit inflammation, and its effect is slightly better than that of the Se-AKPs group, the inflammatory cell infiltration is greatly controlled, the crypt structure is effectively protected or repaired, and the number of goblet cells is restored well. Overall, these results show that Se-AKPs have a protective effect on colitis mice.

[0144] Example 3 Purification and sequence identification of Antarctic krill selenium-chelated peptides

[0145] The prepared Se-AKPs were subjected to ultrafiltration separation. The Antarctic krill selenium-chelated peptides were subjected to fractionation in sequence using ultrafiltration centrifuge tubes with molecular weight cut-offs of 10 kDa and 3 kDa. The ultrafiltration separation conditions were 6000 g / min, centrifugation at 4°C for 20 min, and three components (Se-AKP-I: >10 KDa, Se-AKP-II: 3-10 kDa, and Se-AKP-III: <3 kDa) were obtained by ultrafiltration analysis of Se-AKPs. The anti-inflammatory activity was determined, and the bioactive part was used for the next step.

[0146] LC-MS / MS was used to identify the peptide sequences in the components with higher anti-inflammatory potential. The liquid chromatography conditions were as follows: C18 analytical column (75 um x 150 mm, 3 μm), flow rate of 300 nL / min. Mobile phase A was 0.1% formic acid, 2% ACN; mobile phase B was 0.1% formic acid, 80% ACN; eluted with 6~9%B for 8 min, 9~14%B for 14 min, 14~30%B for 36 min, 30~40%B for 15 min, 40~95%B for 3 min, and 95%B for 5 min. The mass spectrometry conditions were as follows: MSAS scan range (m / z) 100~1500, AGCtarget: 3e6; resolution: 70000 or the like. The MaxQuant software was used to search the mass spectrometry data in the Uniport database. The identified peptide sequences were subjected to bioinformatics analysis to determine their potential anti-inflammatory capacity. The bioactive fractions were lyophilized and used for the next step of functional verification.

[0147] The results of the effect of ultrafiltration components on the production of NO by LPS-stimulated RAW264.7 cells are shown in Figure 19 As shown in Figure 2, the release of NO by LPS-stimulated RAW264.7 macrophages was significantly increased compared with the Control. This indicates that the LPS-induced inflammation model was successfully established. Compared with the model group, the three components: Se-AKPs (MW>10 kDa), Se-AKPs (3 kDa

[0148] After analysis, the sequences of the active peptides from Euphausia superba were LFP, FDL, and FSL Figure 20 .

[0149] Test Example 4: Activity identification of active peptides from Euphausia superba

[0150] The sequences of the active peptides from Euphausia superba, LFP, FDL, and FSL, were selected for chemical synthesis for subsequent experiments to determine their anti-inflammatory activity and mechanism of action. The polypeptides LFP, FDL, and FSL were used to replace the selenium-chelated peptides from Euphausia superba in Test Example 3 to repeat the experimental procedures in (3) and (4) of Test Example 3.

[0151] The results of the effect of active peptides from Euphausia superba on the viability of RAW264.7 cells are shown in Figure 21Compared with the Control group, the LFP, FDL and FSL polypeptide sequences can significantly improve cell viability, in addition, the composite peptide segments LFP+FDL, LFP+FSL and FDL+FSL can also have a proliferation effect on cell growth, and the effect of the composite peptide segment on cell viability is significantly improved compared with the single peptide segment.

[0152] The results of the effect of Antarctic krill active peptide on the NO release amount of RAW264.7 cells are shown in Table 4. Figure 22 Compared with the Control group, the NO release amount of the cells treated with LPS and polypeptides is increased to different limits, and the Model group significantly increases the release amount of cell NO after being induced by LPS, which indicates that the inflammatory model is successfully established. Compared with the Model group, LFP, FDL and FSL have a significant inhibitory effect. The composite peptide segments LFP+FDL, LFP+FSL and FDL+FSL can inhibit the release of NO, and the release amount of NO is significantly reduced compared with the single peptide segment. The results show that the Antarctic krill selenium chelating peptide has good anti-inflammatory activity.

[0153] The above is only an embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. The present application can have various changes and variations for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of Antarctic krill bioactive peptides in the preparation of anti-inflammatory products, characterized in that, The sequence of the Antarctic krill bioactive peptide is FDL.

2. The application according to claim 1, characterized in that, The Antarctic krill bioactive peptides also include bioactive peptides with sequences of LFP or FSL.

3. The application of Antarctic krill selenium chelate peptides in the preparation of anti-inflammatory products, characterized in that... The sequence of the Antarctic krill bioactive peptide is FDL.

4. The application according to claim 3, characterized in that, The Antarctic krill bioactive peptides also include bioactive peptides with sequences of LFP or FSL.

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