Antarctic krill active peptide as well as preparation method and application thereof

Abstract

The invention relates to the field of polypeptides, in particular to an Antarctic krill 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 Antarctic krill active peptide has a good anti-inflammatory effect and can be used for preparing the Antarctic krill selenium chelating peptide, and the prepared Antarctic krill selenium chelating peptide has a good effect in the aspect of 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, and the content is specifically as follows: 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.

[0007] 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.

[0008] Further, the protease in the enzymatic hydrolysis includes at least one of acid protease, neutral protease, papain, alkaline protease, trypsin and pepsin.

[0009] In a specific embodiment of the application, the protease is alkaline protease.

[0010] In a third aspect of the application, the Antarctic krill active peptide is applied in any of the following aspects: A1. application in preparing a product for inhibiting NO release; A2. application in preparing an anti-inflammatory product; A3. Use of the Euphausia superba metal-chelating peptide in the preparation of Euphausia superba metal-chelating peptide.

[0011] Further, the Euphausia superba metal-chelating peptide in A3 comprises 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.

[0012] In a specific embodiment of the present application, the Euphausia superba metal-chelating peptide is Euphausia superba selenium-chelating peptide.

[0013] In a fourth aspect of the present application, a Euphausia superba selenium-chelating peptide is provided, wherein the Euphausia superba selenium-chelating peptide comprises at least one of the Euphausia superba selenium-chelating peptide prepared by using the Euphausia superba active peptide.

[0014] In a fifth aspect of the present application, a method for preparing the Euphausia superba selenium-chelating peptide is provided, wherein the method comprises at least the following steps: 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; S2. adding Na2SeO3 to the Euphausia superba proteolysis solution at a ratio (v / v) of 2:1 to 1:3 to obtain the Euphausia superba selenium-chelating peptide.

[0015] Further, the activity of the protease is between 50 k and 2000 k U / g.

[0016] Further, the method further comprises the step of homogenizing the Euphausia superba shrimp meat and centrifuging to extract the Euphausia superba protein.

[0017] Further, the step of homogenizing the Euphausia superba shrimp meat and centrifuging to extract the Euphausia superba protein is specifically performed as follows: The Euphausia superba shrimp meat is weighed and homogenized with deionized water at a liquid-to-material ratio of 2.5 to 3.5 mL / g. Then, the pH is adjusted to 11 to 12, and then centrifuged to obtain the supernatant. Optionally, the extraction step can be repeated 3 to 5 times. Finally, the supernatants are combined, the pH of the collected supernatant is adjusted to 4.5, and centrifuged again to collect the precipitate and freeze-dried for storage.

[0018] In a sixth aspect of the present application, the Euphausia superba selenium-chelating peptide is used in any of the following aspects: B1. Use in the preparation of a product for inhibiting the release of NO; B2. Use in the preparation of an anti-inflammatory product.

[0019] Further, the Euphausia superba selenium-chelating peptide is used in the preparation of a drug for treating ulcerative colitis.

[0020] The beneficial effects of the present application include but are not limited to: 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 all significantly inhibit the release of NO, and the NO release amount is obviously reduced compared with that of a single peptide segment.

[0021] In the present study, selenium-chelated peptides (Se-AKPs) were successfully prepared from Euphausia superba, and their significant anti-inflammatory activity was confirmed. In vitro experiments showed that Se-AKPs could effectively inhibit the release of NO from LPS-induced RAW264.7 cells. 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

[0022] 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: 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Figure 5 is the CD spectrum (Figure A) and secondary structure distribution diagram (Figure B) of AKPs and Se-AKPs in the embodiments of the present application.

[0027] Figure 6 is the particle size distribution curve (Figure A) and Zate potential structure diagram (Figure B) of AKPs and Se-AKPs in the embodiments of the present application.

[0028] Figure 7are SEM micrographs of AKPs (Figure A) and Se-AKPs (Figure B) in the embodiments of the present application.

[0029] Figure 8 are the results of the influence of different concentrations of LPS on cell viability in the embodiments of the present application.

[0030] Figure 9 are the results of the influence of different concentrations of LPS on NO release in the embodiments of the present application.

[0031] Figure 10 are the results of the influence of different concentrations of AKPs and Se-AKPs on the viability of mouse macrophage RAW264.7 cells in the embodiments of the present application, Figure A is the result of the influence of AKPs on the viability of mouse macrophage RAW264.7 cells, and Figure B is the result of the influence of Se-AKPs on the viability of mouse macrophage RAW264.7 cells.

[0032] Figure 11 are the results of the influence of different concentrations of AKPs and Se-AKPs on the toxicity of LPS-induced RAW264.7 cells in the embodiments of the present application, Figure A is the result of the influence of AKPs on the toxicity of LPS-induced RAW264.7 cells, and Figure B is the result of the influence of Se-AKPs on the toxicity of LPS-induced RAW264.7 cells.

[0033] Figure 12 are the results of the influence of different concentrations of AKPs (Figure A) and Se-AKPs (Figure B) on the release of NO from LPS-induced RAW264.7 cells in the embodiments of the present application.

[0034] Figure 13 are the results of the changes in the disease activity index (DAI) of each group of mice after DSS induction in the embodiments of the present application.

[0035] Figure 14 are the results of the daily weight changes of mice in each experimental group in the embodiments of the present application.

[0036] Figure 15 are photographs of the fecal consistency and fecal occult blood of mice in the Control, DSS, AKPs, Se-AKPs, and DSS+drug groups in the embodiments of the present application.

[0037] Figure 16 are the results of the colon length of mice in the Control, DSS, AKPs, Se-AKPs, and DSS+drug groups in the embodiments of the present application, Figure A is a photograph of the colon of mice, and Figure B is a statistical graph.

[0038] Figure 17Figure A is a level result diagram of IL-6 in serum in the embodiment of the present application, Figure B is a level result diagram of IL-10 in serum in the embodiment of the present application, Figure C is a level result diagram of IL-1β in serum in the embodiment of the present application, Figure D is a level result diagram of TNF-α in serum in the embodiment of the present application, and Figure E is a level result diagram of CPR in serum in the embodiment of the present application.

[0039] Figure 18 Figure is a staining diagram of the influence of Se-AKPs on the pathology of mouse colon in the embodiment of the present application.

[0040] Figure 19 Figure is an anti-inflammatory activity result diagram of Antarctic krill selenium-chelated peptides with different molecular weights in the embodiment of the present application.

[0041] Figure 20 Figure is a secondary mass spectrum diagram of Antarctic krill active peptides FDL (Figure A), FSL (Figure B), and LFP (Figure C) in the embodiment of the present application.

[0042] Figure 21 Figure is an influence result diagram of Antarctic krill active peptides LFP, FDL, and FSL on the activity of RAW264.7 cells in the embodiment of the present application, wherein Figure A is an influence result diagram of Antarctic krill active peptides LFP, FDL, and FSL single components on the activity of RAW264.7 cells, and Figure B is an influence result diagram of Antarctic krill active peptides LFP, FDL, and FSL two-by-two combinations on the activity of RAW264.7 cells.

[0043] Figure 22 Figure is an influence result diagram of 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 Figure A is an influence result diagram of Antarctic krill active peptides LFP, FDL, and FSL single components on the NO release amount of RAW264.7 cells, and Figure B is an influence result diagram of Antarctic krill active peptides LFP, FDL, and FSL two-by-two combinations on the NO release amount of RAW264.7 cells. DETAILED DESCRIPTION

[0044] The present application will be described in detail below with reference to examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are all purchased through commercial channels.

[0045] Example 1 Preparation of Antarctic krill active peptides Euphausia superba meat was weighed into a beaker and homogenized with 3.17 mL / g of deionized water (4 °C). Subsequently, the pH was adjusted to 11.38 with 2 mol / L sodium hydroxide, and after 0.5 h, the supernatant was obtained by centrifugation at 10,000 g for 10 min at 4 °C. 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 1.0 h, the precipitate was collected by centrifugation at 10,000 g for 10 min at 4 °C and stored at -20 °C after freeze-drying. The protein content was determined, and the protein recovery rate was calculated.

[0046] Euphausia superba protein was weighed and dissolved in deionized water at a ratio of 2% (w / v). Six different proteases at a concentration of 2% (w / v) were added to the solution, and the entire enzymatic reaction was carried out in a shaking water bath incubator. The hydrolysis time was 4 h for each enzyme to obtain active peptides from Euphausia superba (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.

[0047]

[0048]

[0049] The degree of hydrolysis of Euphausia superba protein by the six proteases under optimal reaction conditions was calculated as follows: After the enzymatic reaction, the supernatant was centrifuged and immediately subjected to degree of hydrolysis determination. The degree of hydrolysis of Euphausia superba protein was determined using the o-phthaldialdehyde (OPA) method. The specific method is as follows: 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. 40 μL of Euphausia superba enzyme hydrolysate was mixed with 4 mL of OPA reagent at room temperature for 2 min, and the absorbance at 340 nm was measured. Euphausia superba 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 Euphausia superba protein.

[0050] The degree of hydrolysis of Euphausia superba protein was calculated according to the following formula: Degree of hydrolysis (%) = [(NH2) t - (NH2) t0 )] / (NH2)T wherein: (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.

[0051] 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, the two enzymes have more enzyme cleavage sites, and the protein is more easily hydrolyzed into small molecule peptides. Compared with other five proteases, alkaline protease has the best effect.

[0052] Example 2 Preparation of Antarctic krill selenium-chelated peptides Antarctic krill selenium-chelated peptides (Se-AKPs) were prepared by adding 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 centrifuged. The supernatant was taken and 5 times the volume of 95% ethanol solution was added. After mixing uniformly, the mixture was left to stand for 12 h, and then centrifuged to collect the precipitate. 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.

[0053] Test Example 1 Determination of chelation rate of Antarctic krill selenium-chelated peptides This method uses atomic fluorescence spectrometer to accurately determine the selenium chelation rate by hydride generation-atomic fluorescence spectrometry (HG-AFS). First, the chelate was taken in equal amount, digested by microwave, and then diluted with 5% HCl to obtain total selenium solution. The digestion solution needs to be added with 6 mol / L HCl and reduced at 95 ℃ water bath for 30 min, so that Se 6+ is converted to Se 4+ to optimize the hydrogenation efficiency. Then HG-AFS detection (excitation wavelength 196 nm, atomizer temperature 200 ℃, carrier gas argon flow rate 400 mL / min) was used, with 0.8% KBH4 (containing 0.5% NaOH) as reducing agent and 5% HCl as carrier liquid. The free selenium and total selenium concentrations were quantified by selenium standard curve (0-20 μg / L, R 2 >0.995), and the formula is as follows: Chelation rate (%) = (1 - free selenium concentration / total selenium concentration) x 100% The whole experiment was set up with standard addition recovery (85-115%) and parallel determination (RSD < 5%), and the accuracy was verified by NIST SRM 1549 standard material. The detection limit of the method was 0.1 μg / L, which could accurately analyze the binding efficiency of active selenium in selenium peptide complex.

[0054] The results are shown in Figure 2 The polypeptides after enzymolysis by six enzymes (neutral protease, alkaline protease, trypsin, pepsin, acid protease, and papain) were combined with selenium under optimal conditions and the chelation rate was determined. Figure 2 It can be seen that the chelation ability of alkaline protease is higher than that of other conditions, and the chelation rate is 41.8%. Therefore, alkaline protease is selected for chelation of Antarctic krill protein. Alkaline protease has the highest activity under alkaline conditions. Antarctic krill protein may be more easily enzymolyzed under this pH environment to generate a large number of small peptide segments with specific sequences and structures (such as rich in -His, -Lys, -Glu, etc. amino acids), and the functional groups on these peptide segments are more easily combined with selenium ions (Se 4+ ) to form stable chelates. Therefore, Antarctic krill selenium chelated peptides prepared from Antarctic krill active peptides after alkaline protease enzymolysis are selected for subsequent experiments.

[0055] Test Example 2 Physicochemical property detection of Antarctic krill selenium chelated peptides (1) Ultraviolet-visible absorption spectrum The freeze-dried AKPs and Se-AKPs powders were dissolved in deionized water at a concentration of 0.1 mg / mL. The ultraviolet-visible spectrophotometer (Perkin Elmer, Salem, MA) recorded the ultraviolet-visible spectrum of AKPs and Se-AKPs in the wavelength range of 200-600 nm.

[0056] It can be seen from Figure 3 that the absorption peak of AKPs at 270 nm is the strongest, while the absorption peak of Se-AKPs is red-shifted to 280 nm, and the absorption peak intensity increases. The migration of the peak and the change of the peak intensity can be used as a direct marker for the successful chelation of selenium in Antarctic krill protein, and the chelation site is most likely to act on sulfur-containing groups such as cysteine.

[0057] (2) Fluorescence spectrum The freeze-dried AKPs and Se-AKPs were dissolved in deionized water at a concentration of 0.1 mg / mL. Under the excitation wavelength of 288 nm, the fluorescence intensity of AKPs and Se-AKPs was measured at 290-500 nm.

[0058] The fluorescence spectra of AKPs and Se-AKPs in the wavelength range of 290-500 nm are as follows:Figure 4 The results showed that the fluorescence intensity of AKPs decreased significantly after the addition of Se, which was due to the folding and aggregation of some chromogenic amino acids (such as tyrosine and tryptophan) in the peptide during the chelation process. Moreover, the addition of mineral ions could also cause the fluorescence quenching of the sample.

[0059] (3) Circular dichroism (CD) AKPs and Se-AKPs at 1 mg / mL were placed in a quartz cuvette with an optical path of 0.1 cm, and a Jasco J-1500 circular dichroism spectrometer was used to scan 3 times at a speed of 1 nm / s at a wavelength of 190-260 nm. The scanning temperature was (25±0.2) °C.

[0060] The results, as shown in Figure 5 AKPs showed a strong negative peak at 210 nm, indicating that AKPs had an ordered β-sheet structure. The absorbance quickly rose after the 210 nm valley, suggesting the presence of a small amount of α-helix or random coil superposition. Se-AKPs had an absorbance close to 0 at 210 nm, and the overall curve was flat, indicating that selenium chelation significantly reduced the β-sheet structure and converted it to disorder or random coil. Selenium chelation induced the transformation of AKPs from an ordered secondary structure to a random coil dominated flexible conformation by destroying the ordered secondary structure of AKPs. This structural rearrangement may enhance the molecular dynamics and exposure of antioxidant groups, thereby improving its biological activity.

[0061] (4) Particle size / Zeta potential analysis AKPs and Se-AKPs were dissolved in ultrapure water to prepare a solution with a concentration of 1 mg / mL, then added to a U-shaped sample cell, and the particle size and Zate potential of AKPs and Se-AKPs were determined at 25 °C for 5 s using a Zetasizer Nano ZS90 nanoparticle size potential analyzer.

[0062] The results, as shown in Figure 6 The average particle sizes of AKPs and Se-AKPs were 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 chelation of Se or metal ions with the peptide chain at the end of the peptide chain, causing the peptide chain to grow, and structural folding and aggregation reactions, resulting in 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.

[0063] The zeta potentials of AKPs and Se-AKPs were 6.93 mV and -35 mV, respectively. The zeta potential value of Se-AKPs was significantly reduced, which might be due to the electron transfer during the reaction process. Se 2+ AKPs chelated with Se 2+ After being chelated with Se 2+ AKPs, the more electronegative groups were inside, while the positively charged groups were on the outside due to Se 2+ repulsion, 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.

[0064] (5) Scanning electron microscopy (SEM) A certain amount of completely dried AKPs and Se-AKPs chelate powder was adhered to the sample stage with conductive glue, and after being lightly blown with an ear bulb, ion sputtering gold plating film was used, and observation and photography were performed under a field emission scanning electron microscope.

[0065] The results, as shown in Figure 7 , selenium chelation caused a change in the microstructure of Antarctic krill active peptides from interconnected, irregular granular structures to porous, amorphous, highly irregular sponge-like structures by destroying the hydrogen bond network of the peptides and introducing molecular cross-linking. This change in morphology is due to the destruction of the hydrogen bond network of the peptide chain by selenium atoms, inducing molecular cross-linking and disorder, exposing hydrophobic groups, increasing the specific surface area and accessibility of active sites, indicating that selenium chelation reshaped the microstructure-activity relationship of the peptides.

[0066] Test Example 3 In vitro Anti-inflammatory Experiment of Antarctic Krill Selenium Chelated Peptides (1) Cell culture Mouse macrophage RAW264.7 cells were cultured in DMEM high-sugar complete medium (containing 10% fetal bovine serum and 0.1 mg / mL streptomycin and 0.1 mg / mL penicillin). The cells were cultured in a constant temperature 37°C incubator environment containing 95% humidity and 5% carbon dioxide, and were cultured to the logarithmic phase for experiments.

[0067] (2) Screening of LPS concentration Cells in the logarithmic phase were seeded at 5×10 4The density of 1×104 cells / well was inoculated in 96-well plates, 100 μL per well. The culture plates were placed in an incubator for overnight culture, and after the cells adhered (usually 12-24 h), the old liquid of the culture plates was discarded, and then 100 μL of LPS with different concentrations (0, 0.1, 0.5, 1, 1.5 μg / mL) was added to each well for 24 h stimulation. Then the cell viability and NO content were determined.

[0068] The results of cell viability determination are shown in Figure 8 When the concentration of LPS was 0.1 μg / mL, 0.5 μg / mL, and 1 μg / mL, the cell viability showed different degrees of increase, proving that LPS had no toxic effect on RAW264.7 macrophages in this range. When the concentration of LPS was 1.5 μg / mL, the cell viability was 72.9%, at which time LPS had a certain toxic effect on RAW264.7 macrophages.

[0069] The results of NO content are shown in Figure 9 When RAW264.7 macrophages were stimulated with different concentrations of LPS for 24 h, it was found that when the concentration of LPS was 1-1.5 μg / mL, the release of NO was significantly increased. Considering comprehensively, 1 μg / mL of LPS was selected as the concentration for establishing the cell inflammation model.

[0070] (3) Determination of cell viability CCK-8 was used to determine the viability of cells. The cells in the logarithmic phase were trypsinized to detach, and the cells were diluted to a density of 5×10 4 cells / mL using DEME high-glucose complete medium, and inoculated into 96-well culture plates at 100 μL per well. After standing for about 5 minutes, the culture bottles were placed in a cell incubator overnight after the cells were settled. After the cells adhered (12-24 hours), the old culture medium was discarded, and the 96-well plates were gently washed twice with 37°C PBS buffer. 100 μL of AKPs and Se-AKPs with different concentrations (0, 0.1, 0.3, 0.5, 1, 5 mg / mL) were added, respectively, and cultured for 24 h. After 24 h, 10 μL of CCK-8 solution was added to each well, and after re-incubation in the incubator for 1 h, the absorbance (OD 450 ) at 450 nm was measured, and the cell viability was calculated according to the following formula: Cell viability (%) = [A (with drug) - A (blank)] / [A (0 with drug) - A (blank)] x 100% A (drug): absorbance of the well with cells, CCK-8 solution and drug solution; A (blank): absorbance of the well with culture medium and CCK-8 solution without cells; A (0 drug): absorbance of the well with cells and CCK-8 solution without drug solution.

[0071] As shown in Figure 10 , all AKPs had no toxic side effects on RAW264.7 macrophages at a concentration of 0.5-5 mg / mL. Se-AKPs had no toxicity to RAW264.7 macrophages at a concentration of 0.1 mg / mL, and played a certain role in cell proliferation. In addition, other Se-AKPs had significant toxic effects on RAW264.7 macrophages (p<0.05) at a concentration of 0.3-5 mg / mL with increasing concentrations of enzyme hydrolysate. Therefore, the concentration of Se-AKPs was selected as 0.1 mg / mL in the subsequent experiments.

[0072] To determine the safe concentration range of Se-AKPs on RAW264.7 cells induced by 1 μg / mL LPS, the results are shown in Figure 11 , compared with the control group (0 mg / mL), LPS had no effect on the survival rate of mouse macrophage RAW264.7. In addition, different AKPs had no effect on LPS-induced RAW264.7 macrophages at a concentration of 0.1-5 mg / mL, indicating that the concentration range of 5 mg / mL was a safe and effective range. Se-AKPs had no toxicity to LPS-induced RAW264.7 macrophages at 0.1 mg / mL, and played a certain role in cell proliferation. In summary, 0.1 mg / mL was selected as the safe concentration.

[0073] (4) Determination of nitric oxide (NO) content The content of NO was detected by Griess method. Cells in the logarithmic phase were diluted to 5×10 4The density of 1 x 104cells / well was inoculated in 96-well plates, 100 μL per well. The culture plates were placed in an incubator for overnight culture, and after the cells adhered (usually 12-24 h), the old liquid in the culture plates was discarded, and then 100 μL of LPS and phycocyanin enzymatic hydrolysate 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, Griess Reagent I and II were restored to room temperature. According to the sample type, the corresponding diluent was selected to prepare the NO standard product gradient. In a 96-well plate, 50 μL of standard product and treated sample was added to each well. Then, 50 μL of Griess Reagent I and II was added in turn, mixed, and then the absorbance was measured at 540 nm. Using sodium nitrite (NaNO2) standard product, a standard curve (0-100 μmol / L) was drawn, and the content of NO was calculated according to the standard curve.

[0074] The results are shown in Figure 12 As shown in Figure 12 , compared with the blank group (0 mg / mL), the NO release amount of RAW264.7 macrophages stimulated by LPS increased significantly (p<0.05), indicating that the LPS-induced inflammation model was successfully established. Both AKPs and Se-AKPs reduced the NO release amount and enhanced the anti-inflammatory effect with the increase of concentration. However, the NO release amount of Se-AKPs was very low at 0.3-5 mg / mL, which may be because Se-AKPs had a significant toxic effect on RAW264.7 macrophages at 0.3-5 mg / mL. In summary, 0.1 mg / mL was selected as the safe concentration.

[0075] Test 4 Antarctic krill selenium-chelated peptide animal experiment (1) Establishment of animal model The mice were placed in an SPF environment with an independent ventilation system, a 12-hour light-dark cycle, a relative humidity of 55±5%, and food and water provided freely. After one week of adaptation, the mice were housed in cages, and the mouse cages were disinfected and the bedding was replaced three times a week. The experimental animals were divided into 5 groups, with 6 mice in each group. Different drugs were treated by gavage, and the mice were treated at the same time every day. The experimental groups were as follows: Control group (Control group): free drinking of sterile distilled water for 7 d, and gavage of sterile distilled water at the same time; DSS group (DSS group): free drinking of 3% DSS solution to construct ulcerative colitis model for 7 d, and gavage of sterile distilled water at the same time; DSS+AKPs group: free drinking of 3% DSS solution to construct ulcerative colitis model for 7 d, and gavage of AKPs at the same time; DSS+Se-AKPs group: freely drinking 3% DSS solution to build ulcerative colitis model for 7 days, while Se-AKPs; DSS+drug group (Positive control): freely drinking 3% DSS solution to build ulcerative colitis model for 7 days, while gavage with mesalazine enteric-coated tablets.

[0076] The body weight, fecal bleeding, fecal consistency, disease activity index of mice were recorded, so as to evaluate the severity of colitis. Finally, after continuous gavage for seven days and fasting for one day, all mice were euthanized on the eighth day. Serum, colon tissue, cecum, etc. were collected for subsequent analysis.

[0077] (2) Disease activity index 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 of the mice.

[0078] Table 2 DAI score table

[0079] (3) Mouse dissection and tissue collection After the end of 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℃ 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, washed gently with a physiological saline buffer, and dried with filter paper. 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 liquid nitrogen and stored at -80℃ for later use.

[0080] (4) Evaluation of mouse serum cytokines The levels of inflammatory factors interleukin-10 (Interleukin-10, IL-10), tumor necrosis factor-α (Tumornecrosis factor-α, TNF-α), interleukin-1β (Interleukin-1β, IL-1β), interleukin-6 (Interleukin-6, IL-6), C-reactive protein (C-Reactive Protein) in serum were detected by corresponding enzyme-linked immunosorbent assay (ELISA) kit according to the instructions of the manufacturer's manual.

[0081] (5) H&E staining Colonic tissue was dehydrated using a gradient of ethanol solutions of varying concentrations, cleared with ethanol and xylene, embedded in paraffin, sectioned (3 μm), and baked. The sections were stained with hematoxylin and then 0.5% eosin, and mounted with neutral resin. The structures were observed and photographed under a microscope, and histological scoring of colitis was performed, along with the determination of crypt depth.

[0082] The results are as follows: (1) Disease activity index in mice The results are as follows Figure 13 As shown, the study observed the mice's mental state, rectal bleeding, and fecal consistency. During the experiment, the control group mice were in good mental condition, active, and their stool was normal and granular. Compared to the control group, the DSS group mice began to show lethargy, mild diarrhea, and loose stools on the fourth day of DSS treatment. By the eighth day, the DSS group mice were listless, curled up, and exhibited varying degrees of watery bloody stools and anal bleeding. Compared to the DSS group, the drug group, AKPs group, and Se-AKPs group alleviated the above symptoms induced by DSS. Therefore, a comprehensive evaluation of the mouse Disease Activity Index (DAI) showed that the AKPs group and Se-AKPs group could alleviate the decrease in DAI scores caused by DSS.

[0083] (2) Daily weight changes in mice The results are as follows Figure 14 As shown in Figure 14, ulcerative colitis (UC) in mice was induced using sodium dextran sulfate (DSS). All C57BL / 6J mice were randomly assigned to the UC group after a 7-day acclimatization period, and then given 3% DSS for 7 days. The effects of the drug group, AKPs group, and Se-AKPs group on UC mice were observed. Mouse weight changes were monitored throughout the experiment. The results are shown in Figure 14. The weight of mice in the normal control group showed a slow increase during the experiment. Compared to the control group, the weight of the DSS model group began to decrease on the fourth day of DSS modeling. With the extension of the experiment, the weight of mice in the DSS group, drug group, AKPs group, and Se-AKPs group showed an overall decreasing trend, while the drug group, AKPs group, and Se-AKPs group could slow down the DSS-induced weight loss.

[0084] (3) Mouse fecal viscosity and fecal occult blood status according to Figure 15 The five groups of mouse fecal samples and anal region phenotypic characteristics shown clearly reflect the severity of colitis and the intervention effect in each group: Control group: The stool is regular black granular, and the anus is clean without redness or swelling, indicating that the intestines are healthy and there is no inflammation or bleeding.

[0085] DSS group (model group): stool was watery and mixed with obvious fresh red blood, and the anus was red and swollen, which was consistent with the typical characteristics of ulcerative colitis, proving the success of modeling.

[0086] Drug group (Positive Control mesalazine intervention): stool was in blocks and contained 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.

[0087] AKPs group: a small amount of red was visible in the stool, and the anus was red and swollen, reflecting that phosphorus shrimp peptide had certain anti-inflammatory and hemostatic effects, but the effect was weaker than that of the drug group.

[0088] Se-AKPs group: a small amount of red was visible in the stool, but the degree of redness of the anus was lighter than that of the AKPs group, which may be related to the temporary bleeding during the mucosal repair process promoted by selenium.

[0089] (4) Colon length of mice The results, as shown in Figure 16 , colon shortening is one of the typical symptoms of ulcerative colitis. Compared with the control group, DSS caused shortening of the colon, blood appeared in the colon and cecum, and the intestinal contents became thick. AKPs, Se-AKPs, and drug intervention significantly alleviated the shortening of the colon, and could protect against the shortening of the colon caused by DSS.

[0090] (5) Evaluation of serum cytokines The results, as shown in Figure 17 , to further evaluate the effects of Se-AKPs on systemic and intestinal inflammation in colitis mice, we used ELISA to measure the levels of pro-inflammatory cytokines TNF-a, IL-6, IL-1b, CPR and anti-inflammatory cytokine IL-10 in the serum of mice. 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, and 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 reduce the inflammatory state of colitis mice. And Se-AKPs is the most effective.

[0091] (6) Evaluation of serum cytokines The 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 have decreased or disappeared, but still more than the blank group, and the number of goblet cells compared with the model group would have increased to some extent, 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 the introduction of 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.

[0092] Example 3 Purification and sequence identification of Antarctic krill selenium-chelated peptide The prepared Se-AKPs were subjected to ultrafiltration separation. The Antarctic krill selenium-chelated peptides were sequentially fractionated 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 of Se-AKPs were obtained by ultrafiltration analysis (Se-AKP-I: >10KDa, Se-AKP-II: 3-10kDa, and Se-AKP-III: <3kDa). The anti-inflammatory activity was determined, and the bioactive part was used for the next step.

[0093] 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, etc. 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.

[0094] 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 the table, 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

[0095] After analysis, the sequences of the active peptides from Antarctic krill were LFP, FDL, and FSL Figure 20

[0096] Test Example 4: Identification of the activity of active peptides from Antarctic krill The sequences of the LFP, FDL, and FSL active peptides from Antarctic krill were selected for chemical synthesis and subsequent testing to determine their anti-inflammatory activity and mechanism of action. The LFP, FDL, and FSL polypeptides were used to replace the selenium-chelated peptides from Antarctic krill in Test Example 3, and the experimental procedures in (3) and (4) of Test Example 3 were repeated.

[0097] The results of the effect of active peptides from Antarctic krill on the viability of RAW264.7 cells are shown in Figure 21 ​Compared with the Control group, the LFP, FDL and FSL polypeptide sequences can significantly improve the 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 segments on cell viability is obviously improved compared with the single peptide segment.

[0098] The results of the effect of the 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 cell NO release amount is increased to different limits after the cells are treated by LPS and polypeptides, the Model group is significantly increased in the cell NO release amount after being induced by LPS, which indicates that the inflammation model is successfully established. Compared with the Model group, the 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 NO release amount is obviously reduced compared with the single peptide segment. The results show that the Antarctic krill selenium chelating peptide has good anti-inflammatory activity.

[0099] The above only describes the embodiments 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 should be included in the protection scope of the present application.

Claims

1. A Euphausia superba active peptide, characterized in that, The sequence of the active peptide of Euphausia superba includes at least one of LFP, FDL and FSL.

2. A method for preparing the Euphausia superba active peptide according to claim 1, characterized in that, The method includes at least one of solid-phase polypeptide synthesis, liquid-phase polypeptide synthesis and enzymatic hydrolysis.

3. The method of claim 2, wherein, The protease in the enzymatic hydrolysis includes at least one of acid protease, neutral protease, papain, alkaline protease, trypsin and pepsin.

4. The active peptide of Euphausia superba according to claim 1 is applied in any of the following aspects: A1. Application in preparing a product for inhibiting NO release; A2. Application in preparing an anti-inflammatory product; A3. Application in preparing a metal-chelating peptide of Euphausia superba.

5. Use according to claim 4, characterized in that, The metal-chelating peptide of Euphausia superba in A3 includes at least one of ferrous-chelating peptide of Euphausia superba, zinc-chelating peptide of Euphausia superba, selenium-chelating peptide of Euphausia superba and calcium-chelating peptide of Euphausia superba.

6. A selenium-chelating peptide of Euphausia superba, characterized in that, The selenium-chelating peptide of Euphausia superba includes at least the selenium-chelating peptide of Euphausia superba prepared by using the active peptide of Euphausia superba according to claim 1.

7. A method of preparing the Euphausia superba selenium chelate peptide according to claim 6, characterized by, The method includes at least the following steps: S1. Adding at least one of acid protease, neutral protease, papain, alkaline protease, trypsin and pepsin to a solution of Euphausia superba protein to obtain an enzymatic hydrolysate of Euphausia superba protein; S2. Adding Na2SeO3 to the enzymatic hydrolysate of Euphausia superba protein at a ratio of 2:1 to 1:3 (v / v) to obtain a selenium-chelating peptide of Euphausia superba.

8. The method of claim 7, wherein, The method further includes the steps of homogenizing the meat of Euphausia superba and extracting the protein of Euphausia superba by centrifugation.

9. The selenium-chelating peptide of Euphausia superba according to claim 6 is applied in any of the following aspects: B1. Application in preparing a product for inhibiting NO release; B2. Application in preparing an anti-inflammatory product.

10. The selenium-chelating peptide of Euphausia superba according to claim 6 is applied in preparing a medicine for treating ulcerative colitis.

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