Fish by-product active peptide, preparation and application

Abstract

The invention discloses a fish by-product active peptide, a preparation and application, and belongs to the technical field of biological active peptides. The amino acid sequences of the fish by-product active peptide are shown as SEQ ID NO: 1 to SEQ ID NO: 3. Both the single peptide fragment and the compound peptide fragment of the fish by-product active peptide can obviously inhibit NO secretion of a lipopolysaccharide (LPS)-induced macrophage (RAW264.7) inflammation model, so that the fish by-product active peptide has a relatively good application prospect in preparation of a product with an anti-inflammatory effect; and a foundation is laid for high-added-value utilization of salmon byproducts and promotion of research, development and application of functional active peptides.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioactive peptides, and particularly relates to a fish byproduct active peptide, a preparation and application. BACKGROUND

[0002] Salmon is an important economic fish in the world, and a large amount of byproducts (such as fish heads, fish bones, fish skins, etc.) will be produced in the processing process of salmon. These byproducts account for about 40%-50% of the total mass of the fish body, and are usually treated as waste, which not only causes resource waste, but also may cause environmental problems. In recent years, bioactive peptides have become a research hotspot due to their naturalness, safety and diverse physiological activities (such as anti-inflammatory, antioxidant, immunomodulatory, etc.). Anti-inflammatory peptides show great potential in preventing and improving inflammation-related diseases (such as chronic inflammation, autoimmune diseases, etc.). In this context, mining peptide segments with anti-inflammatory activity from salmon byproducts can not only realize the high-value utilization of byproducts, but also provide a theoretical basis and experimental basis for the development of natural anti-inflammatory products. SUMMARY

[0003] In view of the problems in the prior art, the application aims to provide a fish byproduct active peptide, a preparation and application.

[0004] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme: A fish byproduct active peptide, the amino acid sequence of the fish byproduct active peptide is shown in SEQ ID NO: 1-SEQ ID NO: 3.

[0005] The application of the above-mentioned fish byproduct active peptide in the preparation of a product with anti-inflammatory efficacy.

[0006] On the basis of the above-mentioned scheme, the product is food, medicine or health care product.

[0007] On the basis of the above-mentioned scheme, the product further comprises a food, pharmaceutical or health care product acceptable adjuvant.

[0008] A preparation with anti-inflammatory efficacy, the effective component is at least one of YGPV, FSL and LVPF.

[0009] On the basis of the above-mentioned scheme, the concentration of the effective component is 1 mg / mL.

[0010] On the basis of the above-mentioned scheme, the anti-inflammatory efficacy is inhibiting cells from producing NO.

[0011] Advantages of the technical scheme of the application: The application extracts bioactive peptides with anti-inflammatory activity from salmon byproduct through ultrasonic-assisted enzymatic hydrolysis, separates and purifies peptide segments with different molecular weights through ultrafiltration, gel chromatography and liquid chromatography-mass spectrometry, further screens 3 salmon byproduct active peptides with anti-inflammatory efficacy through bioinformatics technology, chemically synthesizes the identified peptide segment sequences, and detects the anti-inflammatory activity of the synthesized single peptide segment and compound peptide segment, so that these bioactive peptides can significantly inhibit the NO secretion of a macrophage (RAW264.7) inflammation model induced by lipopolysaccharide (LPS), and thus have a good application prospect in the preparation of products with anti-inflammatory efficacy, and lay a foundation for the high-value utilization of salmon byproducts and the research and application of functional active peptides. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Effect of different ultrafiltration components on the NO content of LPS-induced RAW264.7 cells; Figure 2 Chromatogram of Sephadex G-15 gel column separation of <3 kDa peptide components; Figure 3 Effect of each purified component on the NO release amount of LPS-induced RAW264.7 cells; Figure 4 Total ion chromatogram of LC-MS / MS; Figure 5 Secondary structure diagram of the peptide segment; Figure 6 Molecular docking visualization diagram of the peptide segments YGPV (a), FSL (b) and LVPF (c); Figure 7 Secondary structure diagram of the three peptide segments FSL; Figure 8 Secondary structure diagram of the three peptide segments LVPF; Figure 9 Secondary structure diagram of the three peptide segments YGPV; Figure 10 Effect of different pretreatment methods and protease types on the degree of hydrolysis of salmon byproduct; Figure 11 Effect of different protease hydrolysates on the survival rate of RAW264.7 cells; Figure 12 Effect of different protease hydrolysates on the cytotoxicity of LPS-induced RAW264.7 cells; Figure 13 Effect of different protease hydrolysates on the NO content of LPS-induced RAW264.7 cells; Figure 14Effects of different concentrations of Al-SAAP on the morphology of LPS-induced RAW264.7 macrophages; Figure 15 Effects of single peptide solutions on the viability (a) and NO content (b) of RAW264.7 cells; Figure 16 Effects of compound peptide solution on the viability (a) and NO content (b) of RAW264.7 cells. Detailed Implementation

[0013] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0014] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0015] In the following embodiments, 1. Method for determining the degree of hydrolysis: During protein hydrolysis, the pH of the solution drops sharply as protons are released. Adding 0.01M NaOH solution helps maintain the pH. The degree of hydrolysis (DH) can be calculated from the volume of alkali added. Calculation formula: C—Concentration of NaOH solution, in mol / L; V — Volume of NaOH solution consumed, mL; a—Dissociation of the amino group, Where pH is the pH value of the hydrolysis system, and pKa is the pKa of the amino group. , where T is the Kelvin temperature; m — Mass of salmon by-product protein in the solution, in g; htot — the total number of peptide bonds in the substrate protein, mmol / g. For salmon by-product protein, htot = 8.6 (calculated based on the amino acid composition of salmon by-product protein).

[0016] 2. Culture of RAW264.7 cells 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 incubator at 37°C with 95% humidity and 5% carbon dioxide until the logarithmic growth phase was reached for experimental use.

[0017] 3. Measurement of cell viability Cell viability was determined using the CCK-8 assay. Cells in the logarithmic growth phase were inoculated at 5 × 10⁻⁶ cells / mL. 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 100 μL of different concentrations of LPS or the test sample (enzyme digest) was added to each well for 24 h of incubation. After incubation, cell morphology was observed and recorded using an inverted microscope. Then, 10 μL of CCK-8 solution was added to each well. After 1 h of incubation, cell viability was calculated by measuring the absorbance at 450 nm using a microplate reader, and the results were calculated using the following formula: Cell viability (%) = [(A) s -A b ) / (A c -A b )]×100 In the formula: A s Indicates the absorbance of the culture medium containing cells, CCK-8, and the sample to be tested; A c This indicates the presence of cell culture medium and CCK-8, with no absorbance of the sample being tested; A b This indicates the absorbance of a culture medium containing CCK-8 that does not contain cells or the sample to be tested.

[0018] 4. Determination of nitric oxide (NO) content NO content was detected using the Griess method. Cells in the logarithmic growth phase were subjected to a reaction at a concentration of 5 × 10⁻⁶ cells / mL. 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 hours), the old culture medium was discarded. Then, 100 μL of different concentrations of LPS or the test sample (enzyme digest) was added to each well and incubated for the specified time. After incubation, the cell culture supernatant was collected, and the NO content was determined using a NO detection kit. In short, 50 μL of supernatant was spread in a 96-well plate, and an equal volume of Griess reagent was added. The absorbance at 540 nm was then measured using a microplate reader. A standard curve (0–100 μmol / L) was plotted using sodium nitrite (NaNO2) standards, and the NO content was calculated based on the standard curve.

[0019] 5. Methods for establishing cellular inflammation models Lipopolysaccharide (LPS) can activate immune cells such as macrophages and induce the release of inflammatory factors (such as TNF-α, IL-6, and IL-1β), making it a classic method for establishing cellular inflammation models. Logarithmically growing RAW264.7 cells were seeded in 96-well plates and cultured at 37°C with 5% CO2 for 12–24 hours until adherence. The old culture medium was discarded, and 1 μg / mL LPS was added, followed by further culturing for 12–24 hours to obtain the cellular inflammation model group (LPS treatment only).

[0020] Example 1 The method for extracting bioactive peptides with anti-inflammatory activity from salmon by-products comprises the following steps: (1) Salmon by-products (fish head, fish bones and fish skin) were minced into no obvious lumps. Distilled water was added at a mass ratio of 1:4 to prepare a salmon by-product solution. The solution was ultrasonically treated at 600 W for 30 min and pre-denatured (passivated) by heating at 90℃ for 10 min.

[0021] (2) Add 3000 U / g of alkaline protease (the amount of protease added per gram of substrate) to the salmon by-product solution and enzymatically hydrolyze for 3 h at 55 °C and pH 10. During this period, the protein solution is continuously stirred and the pH of the system is kept constant with 0.1 M NaOH solution (always maintained at the optimal pH). After the enzymatic hydrolysis is completed, the hydrolysate is placed in a 90 °C water bath for 15 min to inactivate the enzyme. After cooling, it is centrifuged in a high-speed refrigerated centrifuge at 4000 r / min for 20 min at a temperature of 4 °C. The supernatant is filtered and freeze-dried to obtain crude peptide powder of salmon by-product.

[0022] (3) The enzymatically hydrolyzed salmon by-product anti-inflammatory peptide (SAAP) was diluted, filtered first through a 0.45 μm filter membrane, then through a 0.22 μm filter membrane, and poured into a container. Ultrafiltration was performed using membranes with molecular weights of 10 KDa and 3 KDa, with a centrifugal force of 4000 r / min, an operating temperature of 25℃, and times of 15 min and 40 min, respectively. After ultrafiltration, the filtrate was freeze-dried to obtain salmon by-product active peptide powders with molecular weights >10 KDa, 3-10 KDa, and <3 KDa. Sample peptide solutions of 1 mg / mL were prepared, and the anti-inflammatory activity of salmon by-product active peptides with molecular weights >10 KDa, 3-10 KDa, and <3 KDa was determined, with the LPS-treated model group as a control. The results are as follows: Figure 1As shown, LPS treatment of RAW264.7 macrophages for 24 h significantly increased NO release by 9-fold. The three ultrafiltration fractions all significantly reduced NO content compared to the model group, with NO inhibition rates of 14.19%, 7.06%, and 3.20%, respectively (p < 0.05). The < 3 kDa fraction exhibited the strongest anti-inflammatory activity and showed a higher NO inhibition rate compared to the original enzymatic hydrolysate.

[0023] (4) The <3 kDa enzymatic hydrolysate was further purified by filtration on a Superdex™ Increase 10 / 300 GL gel permeation column (10×300 mm). The column was eluted with deionized water (pH 10), and the fractions were collected at a flow rate of 1 mL / min. The fractions were detected at 280 nm. Three polypeptide fractions were collected in the order of peak elution and named F1, F2, and F3, respectively. Figure 2 The inhibitory effects of these three polypeptide components on cell viability and NO content, which are related to anti-inflammatory activity, were measured, with the LPS-treated model group used as a control. Results are as follows: Figure 3 As shown, pretreatment of RAW264.7 macrophages with the three enriched fractions (F1, F2, and F3) significantly reduced NO content (inhibition rates of 5.72%, 2.32%, and 4.41%, respectively). F2 exhibited stronger anti-inflammatory activity than the other two fractions. Furthermore, the NO inhibition rate of this peptide fraction was significantly higher than that of the original enzymatic hydrolysate, indicating improved anti-inflammatory activity.

[0024] (5) The most anti-inflammatory active peptides with a molecular weight <3 kDa obtained above were screened and analyzed by liquid chromatography-mass spectrometry (LC-MS) to identify the structure of the active peptides. The salmon by-product peptide peaks with the most anti-inflammatory activity and a molecular weight <3 kDa were separated by an RSLC C18 column using an Easy-nLC high performance liquid chromatography system. Gradient elution: mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; the mass spectrometry scanning mode was information-dependent acquisition mode, the primary mass spectrometry scanning resolution was 70,000, and a maximum of 20 secondary spectra with charges from 2+ to 5+ were acquired in each working cycle.

[0025] (6) The potential bioactivity of the obtained salmon by-product peptides was predicted and scored (0-1) using the Peptide Ranker program. Peptides with a score >0.5 were selected, and peptides with a toxicity threshold prediction <0.4 were selected using the ToxinPred3.0 website. The secondary structure of the peptides was then predicted and analyzed.

[0026] The results are as follows Figure 4As shown, the anti-inflammatory peptide (SAAP) from salmon byproducts was identified by liquid chromatography-mass spectrometry (LC-MS / MS), yielding a total of 2220 peptides. Of these, 1074 had a molecular weight less than 1000 (MASS); 424 had no peptide modification (PTM); 424 had ALC sequence confidence greater than 50% (ALC); 24 had an area peak area greater than 107 (Area); 16 had a Peptide Ranker score greater than 0.5 (Peptide Ranker score); and 10 had a toxicity prediction threshold less than 0.4 (Total toxicity prediction threshold). The secondary structures of 10 suitable peptides were finally selected as follows: Figure 5 As shown.

[0027] (7) 3D structures were obtained from the PubChem and Protein Data Bank (PDB) databases, and molecular docking screening of peptides and iNOS proteins was performed using AutoDock Vina. The crystal structure of iNOS protein (PDB code: 3NW2) was downloaded from the protein database. Water molecules and ligand structures in the receptor protein iNOS were first removed, and then converted to pdbqt format for storage for AutoDock Vina docking. The grid spacing for AutoDock Vina molecular docking was set to 1 Å, and the number of grid points in the X, Y, and Z directions was set to 30 × 30 × 30. Molecular docking of salmon by-product peptide sequences with anti-inflammatory factors was performed using AutodockTools software to study the binding energy, hydrogen bonds, and intermolecular forces between anti-inflammatory factors (receptors) and peptides (ligand molecules); the results are shown in Table 1.

[0028] Table 1 Binding energies of 10 peptides Table 1 shows the binding energies of each peptide after docking. The larger the absolute value of the binding energy, the better the binding effect between the receptor and ligand. Therefore, YGPV, FSL, and LVPF peptides were selected for subsequent synthesis (Table 2).

[0029] Table 2. Summary of SAAP peptides FSL, LVPF, and YGPV information The docking results of salmon by-product peptides were visualized using Pymol software, and the sequences were then chemically synthesized. Visualizations of the docking of peptides YGPV, FSL, and LVPF are shown below. Figure 6 As shown. The secondary spectra of the three chemically synthesized peptides FSL, LVPF, and YGPV are as follows. Figures 7-9 As shown.

[0030] Example 2 Effects of different pretreatment methods and protease types on the degree of hydrolysis of salmon by-products Salmon by-products (fish heads, bones, and skin) were minced until they were free of obvious lumps. Distilled water was added at a mass ratio of 1:4 to prepare a salmon by-product solution. This solution was divided into two main groups, each containing six subgroups. The first group underwent ultrasonic cell disruption treatment at 600 W for 30 min before enzymatic hydrolysis; the second group did not undergo ultrasonic treatment. Neutral protease, papain, trypsin, alkaline protease, pepsin, and flavor protease (3000 U / g each) were added to the six subgroups within each main group to perform enzymatic hydrolysis. The hydrolysis temperature and pH were maintained at the optimal temperatures and pH levels for each protease (Table 3), and the hydrolysis lasted for 3 h. After hydrolysis, the hydrolysate was placed in a 90℃ water bath for 15 min to inactivate the enzymes. After cooling, it was centrifuged in a high-speed refrigerated centrifuge at 4000 r / min for 20 min, with the temperature controlled at 4℃, to obtain the supernatant. The effects of different pretreatment methods and protease types on the degree of hydrolysis (DH, %) and in vitro anti-inflammatory activity of salmon by-products were determined.

[0031] Table 3. Optimal temperature and pH for different proteases The results are as follows Figure 10 As shown, comparing ultrasound-assisted enzymatic hydrolysis with conventional enzymatic hydrolysis, the ultrasound-assisted enzymatic hydrolysis method with an ultrasound power of 600W and an ultrasound time of 30 minutes generally resulted in a higher degree of protease hydrolysis compared to the conventional method. This is because the cavitation effect of ultrasound allows the salmon by-product proteins to fully bind with the enzyme, promoting the hydrolysis reaction and increasing the degree of hydrolysis. After ultrasound treatment, the salmon by-products hydrolyzed using alkaline protease had the highest degree of hydrolysis, at 12.11%.

[0032] Effects of different concentrations of protease hydrolysate on the survival rate of mouse RAW264.7 macrophages RAW264.7 cells were treated with different protease hydrolysates (SAAP) obtained from the enzymatic hydrolysis of salmon by-products using different proteases. The effect of different concentrations of SAAP (0, 0.1, 0.5, 1, 3, and 5 mg / mL) on cell viability after 24 h of treatment was examined. The results are as follows: Figure 11 As shown, within the concentration range of 0-3 mg / mL, different SAAPs showed no toxic side effects on mouse macrophage RAW264.7 cells with increasing concentration of enzymatic hydrolysate. Among them, alkaline protease hydrolysate (Al-SAAP) showed the best cell survival rate.

[0033] Effects of different protease hydrolysates on LPS-induced RAW264.7 cytotoxicity The effects of different protease hydrolysates (SAAPs) obtained from the enzymatic hydrolysis of salmon by-products by different proteases on the viability of RAW264.7 macrophages were determined using the CCK-8 assay. The results are as follows: Figure 12 As shown, all six protease hydrolysates exhibited no toxicity to LPS-induced RAW264.7 macrophages at concentrations of 0.1-1 mg / mL, and all showed some proliferative effect on cell growth, indicating that the concentration range of 0.1-1 mg / mL is a safe range. In particular, the alkaline protease hydrolysate (Al-SAAP) showed the best effect on cell proliferation. The cell viability at a concentration of 3 mg / mL was 141.73%.

[0034] Effects of different protease hydrolysates on NO content in LPS-induced RAW264.7 cells Different proteases were used to hydrolyze salmon by-products to obtain different protease hydrolysates (SAAP). The effect of different concentrations of SAAP on the NO content of LPS-induced RAW264.7 cells was determined. Logarithmically growing RAW264.7 cells were seeded in 96-well plates and cultured at 37℃ in a 5% CO2 incubator for 12–24 hours. The old culture medium was discarded, and different concentrations of SAAP were added for pre-incubation for 2 h, followed by stimulation with 1 μg / mL LPS for 12–24 h. The NO content of the cell supernatant was then measured after the incubation. Results are shown below. Figure 13 As shown in the figure, compared with the control group, LPS-stimulated RAW264.7 macrophages showed a significant increase in NO release, indicating the successful establishment of the LPS-induced inflammation model. Compared with the model group (LPS-induced), RAW264.7 macrophages treated with SAAP at concentrations of 0.1-3 mg / mL showed a decrease in NO release, indicating that all enzymatic hydrolysates exhibited anti-inflammatory effects. Among them, the decrease in cell NO content after treatment with Al-SAAP was the greatest, showing the best effect.

[0035] Effects of different concentrations of Al-SAAP hydrolysate on LPS-induced morphology of RAW264.7 macrophages Al-SAAP was obtained by enzymatic hydrolysis of salmon byproducts using alkaline protease. The effects of different concentrations of Al-SAAP on the morphology of LPS-induced RAW264.7 macrophages were determined, and the results are as follows: Figure 14 As shown, from Figure 14 As can be seen in (a), normal RAW264.7 macrophages are nearly oval and smooth. Figure 14 In the middle (b), which is the model group, RAW264.7 macrophages induced with 1 μg / mL LPS showed differentiation, increased pseudopodia, and most cells were dendritic. Figure 14In the middle (c) and (d), the experimental groups were treated with different concentrations of Al-SAAP. It can be seen that the cell surface treated with Al-SAAP gradually became smooth and shrank, and the pseudopodia were continuously reduced, indicating that salmon by-product peptides can effectively alleviate inflammation.

[0036] Example 3 A bioactive peptide with anti-inflammatory effects, wherein the amino acid sequence of the bioactive peptide is any one of SEQ ID NO:1 to SEQ ID NO:3.

[0037] SEQ ID NO:1: YGPV; SEQ ID NO:2: FSL; SEQ ID NO:3: LVPF.

[0038] Example 4 A bioactive peptide preparation with anti-inflammatory effects, wherein the active ingredient of the preparation comprises at least one of the bioactive peptides shown in SEQ ID NO:1 to SEQ ID NO:3.

[0039] Using water as a solvent, single peptides (YGPV, FSL, LVPF) were prepared into solutions with a concentration of 1 mg / mL. The anti-inflammatory activity and effect on cell viability of the single peptide solutions were then assessed. Results are as follows: Figure 15 As shown, the order of activity was FSL > LVPF > YGPV. The single peptide FSL exhibited the best anti-inflammatory activity, with a cell viability of 133.14% and a NO content of 5.79%, which was four times lower than the control group.

[0040] Using water as a solvent, YGPV, FSL, and LVPF peptides were combined in pairs to obtain FSL+LVPF, FSL+YGPV, and LVPF+YGPV complex peptide solutions. The mass ratio of the two peptides in the complex peptide solutions was 1:1, and the concentration of each peptide was 1 mg / mL. The anti-inflammatory activity and effect on cell viability of the three complex peptide solutions (FSL+LVPF, FSL+YGPV, and LVPF+YGPV) were detected. The results are as follows: Figure 16 As shown, FSL+LVPF > FSL+YGPV > LVPF+YGPV. The complex peptide FSL+LVPF exhibited the best anti-inflammatory activity, with cell viability at 137.42% and NO content at 3.60%, which was 6 times lower than the control group.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A bioactive peptide derived from fish byproducts, characterized in that, The amino acid sequences of the fish by-product bioactive peptides are shown in SEQ ID NO:1~SEQ ID NO:

3.

2. The use of the fish by-product bioactive peptides of claim 1 in the preparation of products with anti-inflammatory effects.

3. The application of the fish by-product bioactive peptide according to claim 2 in the preparation of products with anti-inflammatory effects, characterized in that, The product is food, medicine, or health supplement.

4. The application of the fish by-product bioactive peptide according to claim 3 in the preparation of products with anti-inflammatory effects, characterized in that, The product further includes food, pharmaceutical or health food-grade excipients.

5. A preparation with anti-inflammatory effects, characterized in that, The active ingredient is at least one of YGPV, FSL, and LVPF.

6. The anti-inflammatory formulation according to claim 5, characterized in that, The concentration of the active ingredient is 1 mg / mL.

7. The formulation with anti-inflammatory effects according to claim 5 or 6, characterized in that, The anti-inflammatory effect described is the inhibition of NO production by cells.

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