Oyster active peptide and application thereof in antioxidation and auxiliary regulation of blood sugar

Abstract

The application discloses oyster active peptides and application thereof in antioxidation and auxiliary regulation of blood sugar, and belongs to the technical field of active peptides. The sequence of the oyster active peptide is PYR, FKP, LPPR, NWP or PRPP. The oyster active peptide has good antioxidation activity and alpha-glucosidase inhibition activity. The oyster active peptide has high safety and effectiveness and is prepared from food-derived proteins. The obtained antioxidant and alpha-glucosidase inhibiting peptide can be applied to functional products including food and medicine as auxiliary antioxidant and hypoglycemic active ingredients, and has important guiding significance for development of new auxiliary antioxidant and hypoglycemic products.

Description

Technical Field

[0001] The invention belongs to the field of active peptides, and in particular relates to oyster active peptides and applications thereof in anti-oxidation and auxiliary blood sugar regulation. Background Art

[0002] As natural antioxidants, antioxidant peptides are easily absorbed, stable, and non-immunoreactive. They offer nutritional benefits, blood pressure lowering, and immune-boosting properties. Due to their natural, safe, non-toxic, and high nutritional value, they are widely used in the food industry and have become one of the most widely used natural active ingredients in functional foods. Developing nutritionally safe antioxidant peptides from low-cost biological resources has long been a research hotspot in the peptide field.

[0003] α-glucosidase inhibitors can effectively regulate blood sugar by inhibiting the activity of α-glucosidase. Supplementing exogenous α-glucosidase inhibitors, such as α-glucosidase inhibitory peptides, inhibits the decomposition of carbohydrates and improves postprandial hyperglycemia, which has become a hot topic in the intervention research of diabetes and its complications in recent years.

[0004] There is a close relationship between antioxidant effects and blood sugar regulation. Antioxidant supplementation can assist in lowering blood sugar by reducing oxidative stress, protecting pancreatic cells, improving insulin sensitivity, and reducing the risk of complications. A healthy lifestyle (such as diet and exercise) in blood sugar lowering management can also enhance antioxidant capacity, and the two form a synergistic effect.

[0005] Oyster( Pacific oyster Oysters (oysters) are a type of mollusk belonging to the family Ostreidae of the order Pinctada margarita, commonly known as sea oysters or oysters. They have an irregular bivalve structure and are characterized by delicious meat rich in nutrients such as protein, zinc, calcium, and taurine. Both their shells and meat are used as medicine. They are a significant marine resource widely distributed across tropical and temperate waters, possessing high therapeutic and medicinal value. Currently, no research has focused on the active oyster peptides found in simulated oyster digestion products.

[0006] Bioinformatics tools and molecular docking-based virtual screening for active peptides are used to identify target peptides based on known amino acid sequences. Using database searches and software analysis, the potential bioactivity, safety, and structure-activity relationship of the peptides are simulated and predicted. Further evaluation of ligand-receptor interactions allows for rapid targeted screening of target active peptides. Compared with traditional preparation methods, bioinformatics tools and molecular docking-based virtual screening can significantly save time and experimental costs. In practice, combining these two approaches can significantly improve screening efficiency and accuracy. This method has been used to screen natural active peptides from oysters, which is of great significance for the development of functional products with antioxidant and blood sugar-regulating properties. Summary of the Invention

[0007] The purpose of the present invention is to provide an oyster active peptide and application thereof.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] The present invention screened out five oyster active peptides with relatively high activity from oysters, and the sequences of the oyster active peptides are PYR (Pro-Tyr-Arg), FKP (Phe-Lys-Pro), LPPR (Lys-Pro-Pro-Arg, as shown in SEQ ID NO.1 in the sequence listing), NWP (Asn-Trp-Pro), and PRPP (Pro-Arg-Pro-Pro, as shown in SEQ ID NO.2 in the sequence listing).

[0010] The above-mentioned method for screening oyster active peptides comprises the following steps:

[0011] (1) Preparation of oyster meat homogenate: Take a certain amount of oysters, wash and dry them, remove all the soft tissue and juice, and homogenize them in a high-speed tissue grinder three times, each time for 30 seconds to obtain a homogenate;

[0012] (2) INFOGEST in vitro static simulated digestion model: First, simulated saliva was added at a volume ratio of 1:1, and digested in a constant temperature oscillator at 37 ℃ and 100 rpm for 2 minutes to simulate oral digestion; then, the pH was adjusted to 3.0 with 6 mol / L hydrochloric acid solution to terminate the oral digestion process, and simulated gastric fluid was added at a volume ratio of 1:1, mixed evenly, and digested at a constant temperature oscillator for 2 hours to simulate gastric digestion. During the experiment, the pH of the digestive fluid was controlled at 3.0 with 1 mol / L hydrochloric acid solution; finally, the pH was adjusted to 6.0 with 5 mol / L sodium hydroxide solution to terminate the gastric digestion process, and simulated intestinal fluid was added at a volume ratio of 1:1, mixed evenly, and then the pH was adjusted to 7.0 with 1 mol / L sodium bicarbonate solution, and digested at a constant temperature oscillator for 2 hours to simulate intestinal digestion;

[0013] (3) Identification of oyster protein peptide sequences: Peptide mapping of oyster protein simulated digestion products was performed using the Thermo Fisher EASY-nLC1200 Q Exactive liquid chromatography-mass spectrometry system;

[0014] (4) Bioinformatics tools assisted the screening of oyster peptides: the novelty of peptides was queried through the BIOPEP database, the biological activity of peptides was predicted through the Peptide Ranker tool, the digestion resistance and cell membrane permeability of peptides were predicted through the Peptide Cutter tool and the CPPpred tool, the stability of peptides in blood was evaluated through the PLiePred tool, and the potential toxicity and physicochemical properties of peptides were predicted through the Toxin Pred tool;

[0015] (5) Molecular docking: The screened antioxidant peptides were used as ligands and Keap1 as receptors, and the action sites and interaction forces between the antioxidant peptides and Keap1 were analyzed by molecular docking technology; the screened α-glucosidase inhibitory peptides were used as ligands and α-glucosidase as receptors, and the action sites and interaction forces between the α-glucosidase inhibitory peptides and α-glucosidase were analyzed by molecular docking technology.

[0016] (6) Determination of antioxidant activity: In vitro antioxidant activity tests were performed, including DPPH method, ABTS method and ORAC method to find the peptide with the strongest antioxidant activity.

[0017] (7) Determination of α-glucosidase inhibitory activity: Peptide solutions with different gradient concentrations and α-glucosidase solution were added to the ELISA plate, mixed and incubated, and then PNPG solution was added for reaction. The colorless PNPG was hydrolyzed by α-glucosidase to release PNP. PNP is yellow under alkaline conditions. The amount of PNP produced at 405 nm was measured to calculate the change in enzyme activity, and peptides with α-glucosidase inhibitory effect were screened out; the PNPG refers to p-nitrophenyl-α-D-pyranoglucoside, and the PNP refers to nitrophenol.

[0018] Preferably, in the operation of step (1), after homogenization, the homogenate needs to be placed in an experimental refrigerator or immediately undergo subsequent experiments to avoid long-term storage at room temperature which may cause degradation of protein and polypeptide components.

[0019] Preferably, in the operation of step (2), after each stage of simulated digestion is completed, if there is no need to proceed to the next stage of simulated digestion, the simulated digestion solution needs to be immediately placed in an ice bath to terminate the digestion reaction of this stage.

[0020] Preferably, in step (3), the components obtained in step (2) are separated using a Nano-HPLC liquid phase system EASY-nLC 1200 (ThermoFisher Scientific) and then subjected to mass spectrometry analysis using a Q-Exactive mass spectrometer (Thermo Fisher Scientific); the obtained mass spectrometry data are analyzed using the software MAXQUANT, and the obtained polypeptide sequence is compared with the Uniprot-Acipenser database to obtain the protein source of the peptide sequence.

[0021] Preferably, in the operation of step (4), the basic screening conditions are that the Peptide Ranker score > 0.6 and the toxicity is absent.

[0022] Preferably, in the operation of step (5), the result of molecular docking is expressed as a binding energy value, and the docking binding energy with Keap1 is less than -8.0 kcal / mol as a screening condition. The docking binding energy with α-glucosidase is less than -6.0 kcal / mol as a screening condition.

[0023] The oyster active peptide of the present invention can be synthesized and produced by solid-phase synthesis methods, including but not limited to the Fmoc-SPPS method, the BOC-SPPS method, and the fragment condensation connection method. All possible synthetic methods can be used to more efficiently obtain the oyster active peptide, thereby providing materials for the further utilization of the screened polypeptides.

[0024] Advantages of the present invention:

[0025] This invention discloses an oyster active peptide and its application. The oyster active peptide was obtained through simulated digestion, LC-MS / MS mass spectrometry identification, bioinformatics analysis and prediction, molecular docking, peptide synthesis, and activity validation screening. A total of 349 sequences were identified through peptide mapping, of which 58 active sequences have been reported. Of the remaining 291 unreported sequences, 85 had a Peptide Ranker score exceeding 0.6. Based on Peptide Ranker scores, stability, digestion resistance, and cell membrane permeability, 17 peptide sequences were selected for molecular docking. Nine peptide sequences showed binding energies with Keap1 below -8.0 kcal / mol, and eight peptide sequences showed binding energies with α-glucosidase below -6.0 kcal / mol. After comprehensive consideration of potential activity, safety, and bioaccessibility, five sequences were selected for synthesis and activity validation. Experimental results demonstrated that the peptides PYR, FKP, LPPR, NWP, and PRPP all exhibited certain antioxidant and α-glucosidase inhibitory activities. The above peptide segments can be added to functional products such as pharmaceutical compositions or nutritional products as auxiliary antioxidant and blood sugar regulating active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a technical roadmap for the preparation of oyster peptides in the embodiments of the present invention;

[0027] Figure 2 This is the overall three-dimensional structure diagram of the docking of peptide PYR and Keap1 molecule;

[0028] Figure 3 A two-dimensional schematic diagram of the interaction between the active site residues of the PYR peptide and the Keap1 molecule;

[0029] Figure 4 This is the overall three-dimensional structure diagram of the docking of peptide FKP and Keap1 molecule;

[0030] Figure 5 A two-dimensional schematic diagram of the interaction between the active site residues of the FKP peptide and the Keap1 molecule;

[0031] Figure 6 This is the overall three-dimensional structure diagram of the docking of peptide LPPR and Keap1 molecule;

[0032] Figure 7 A two-dimensional schematic diagram of the interaction between the active site residues of the LPPR peptide and the Keap1 molecule;

[0033] Figure 8 This is the overall three-dimensional structure diagram of the docking of peptide NWP and Keap1 molecule;

[0034] Figure 9A two-dimensional schematic diagram of the interaction between the active site residues of the peptide NWP and the Keap1 molecule;

[0035] Figure 10 This is the overall three-dimensional structure diagram of the docking of peptide PRPP and Keap1 molecule;

[0036] Figure 11 A two-dimensional schematic diagram of the interaction between the active site residues of the peptide PRPP and the Keap1 molecule;

[0037] Figure 12 This is the overall three-dimensional structure diagram of the docking of polypeptide PYR and α-glucosidase molecule;

[0038] Figure 13 A two-dimensional schematic diagram of the interaction between the active site residues of the PYR peptide and the α-glucosidase molecule;

[0039] Figure 14 This is the overall three-dimensional structure diagram of the docking of polypeptide FKP and α-glucosidase molecule;

[0040] Figure 15 A two-dimensional schematic diagram of the interaction between the active site residues of the FKP peptide and the α-glucosidase molecule;

[0041] Figure 16 This is the overall three-dimensional structure diagram of the docking of the peptide LPPR and the α-glucosidase molecule;

[0042] Figure 17 A two-dimensional schematic diagram of the interaction between the active site residues of the LPPR peptide and the α-glucosidase molecule;

[0043] Figure 18 This is the overall three-dimensional structure diagram of the docking of peptide NWP and α-glucosidase molecule;

[0044] Figure 19 A two-dimensional schematic diagram of the interaction between the active site residues of the NWP peptide and the α-glucosidase molecule;

[0045] Figure 20 This is the overall three-dimensional structure diagram of the docking of polypeptide PRPP and α-glucosidase molecule;

[0046] Figure 21 A two-dimensional schematic diagram of the interaction between the active site residues of the PRPP peptide and the α-glucosidase molecule;

[0047] Figure 22 This is a diagram showing the liquid phase detection results of the synthetic polypeptide PYR in an embodiment of the present invention;

[0048] Figure 23 This is a diagram showing the liquid phase detection results of the synthetic polypeptide FKP in an embodiment of the present invention;

[0049] Figure 24 This is a diagram showing the liquid phase detection results of the synthetic polypeptide LPPR in an embodiment of the present invention;

[0050] Figure 25 This is a diagram showing the liquid phase detection results of the synthetic polypeptide NWP in an embodiment of the present invention;

[0051] Figure 26 This is a diagram showing the liquid phase detection results of the synthetic polypeptide PRPP in an embodiment of the present invention;

[0052] Figure 27 This is a diagram showing the mass spectrometry results of the synthetic polypeptide PYR according to an embodiment of the present invention;

[0053] Figure 28 This is a diagram showing the mass spectrometry results of the synthetic polypeptide FKP in an embodiment of the present invention;

[0054] Figure 29 Graph showing the mass spectrometry results of the synthetic polypeptide LPPR according to an embodiment of the present invention;

[0055] Figure 30 This is a diagram showing the mass spectrometry results of the synthetic polypeptide NWP according to an embodiment of the present invention;

[0056] Figure 31 1 is a diagram showing the mass spectrometry detection results of the synthetic polypeptide PRPP according to an embodiment of the present invention;

[0057] Figure 32 This is a graph showing the effect of the synthetic peptide on the DPPH free radical scavenging activity in an embodiment of the present invention;

[0058] Figure 33 This is a graph showing the effect of the synthetic peptide on the ABTS free radical scavenging activity in an embodiment of the present invention;

[0059] Figure 34 This is a graph showing the effect of synthetic peptides on ORAC activity in an embodiment of the present invention;

[0060] Figure 35 This is a graph showing the effect of the synthetic peptide on the α-glucosidase inhibitory activity in an example of the present invention. DETAILED DESCRIPTION

[0061] The present invention will be further described below in conjunction with specific examples. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.

[0062] Example 1: Screening of polypeptides

[0063] A technical route for preparing oyster peptides is as follows Figure 1 As shown, the in vitro simulated digestion of oysters represents the digestion of oyster meat using the INFOGEST in vitro static simulated digestion model.

[0064] The specific steps include:

[0065] (1) Digestion of oyster meat based on the INFOGEST in vitro static simulated digestion model: First, a certain amount of oysters was washed and dried, and all the soft tissues and juices were removed. The oysters were homogenized in a high-speed tissue grinder three times, each time for 30 seconds, to obtain a homogenate. Secondly, simulated saliva (α-amylase EC 232-565-6, final concentration 150 U / mL) was added at a volume ratio of 1:1, and digestion was carried out in a constant temperature shaker at 37°C at 100 rpm for 2 min to simulate oral digestion. Then, the pH was adjusted to 3.0 with 6 mol / L hydrochloric acid solution to terminate the oral digestion process, and simulated gastric fluid (pepsin EC 3.4.23.1 and gastric lipase EC 3.1.1.3, final concentrations 4000 U / mL and 120 U / mL, respectively) was added at a volume ratio of 1:1, mixed evenly, and digested at a constant temperature shaker for 2 h to simulate gastric digestion (during the experiment, the pH of the digestive fluid was controlled at around 3.0 with 1 mol / L hydrochloric acid solution). Finally, the pH was adjusted to 6.0 with 5 mol / L sodium hydroxide solution to terminate the gastric digestion process, and simulated intestinal fluid (pancreatin EC3.4.21.4, final concentration 200 U / mL, ox bile salt final concentration 20 The mixture was mixed evenly with 1 mol / L sodium bicarbonate solution, and the pH was adjusted to 7.0. The mixture was digested at a constant temperature with shaking for 2 h to simulate intestinal digestion (during the experiment, the pH of the digestive fluid was controlled at around 7.0).

[0066] (2) LC-MS / MS identification of peptide sequences: Separation was performed using a Nano-HPLC liquid phase system EASY-nLC 1200 (ThermoFisher Scientific) followed by analysis using a Q-Exactive mass spectrometer (Thermo Fisher Scientific). Analysis was performed on a C18 capture column (100 μm inner diameter, 2 cm length, 5-μm C18; SC001, Thermo Fisher Scientific) and a C18 analytical column (75 μm inner diameter, 10 cm length, 3-μm C18; SC2003, Thermo Fisher Scientific). The two mobile phases were buffer A (0.1% formic acid / 99.9% water) and buffer B (80% acetonitrile / 1% formic acid / 19% water). The proportion of buffer B was increased from 4% to 100% over 90 min at a flow rate of 250 nL / min. Scans were performed at m / z 200 at a resolution of 70,000, with a scan range of 400 to 1700 m / z. The 10 most abundant MS1 features were selected and fragmented by high-energy collisional dissociation at m / z 200 at a resolution of 17,500. Ion injection time and ion target values ​​were set to 20 ms, 3E6 (survey scan) and 60 ms, 5E5 (MS / MS scan), respectively. Data were acquired using Xcalibur software (ThermoScientific).

[0067] (3) Bioinformatics tools assisted screening of oyster peptides:

[0068] First, the novelty of the peptides was queried using the BIOPEP database, and sequences with reported activity were not screened further. The Peptide Ranker tool was used to predict the biological activity of the peptides, and sequences with a score of more than 0.6 were considered to have potential activity. The Peptide Cutter tool was used to predict the digestion resistance of the peptides. If the sequence did not contain sites that could be cleaved by pepsin (Pepsin pH 1.3 and pH>2.0, EC 3.4.23.1), trypsin (Trypsin, EC3.4.21.4), and chymotrypsin (Chymotrypsin, EC 3.4.21.1), the sequence was considered to have the potential to resist gastrointestinal digestion. The CPP pred tool was used to predict the cell membrane permeability of the peptides. Sequences with a score of more than 0.15 were considered to have the potential for complete transmembrane absorption. The PLiepred tool was used to evaluate the stability of the peptides in the blood. Peptide sequences with a half-life value greater than 800 s were considered to have a certain stability in the blood. The Pred tool predicts the potential toxicity and physicochemical properties of peptides, and only sequences that are deemed to have no potential toxicity can be subsequently synthesized and verified.

[0069] A total of 349 sequences were obtained through peptide mapping, of which 58 active sequences have been reported. Among the remaining 291 unreported sequences, 85 had a Peptide Ranker score of more than 0.6. Based on the Peptide Ranker score, stability, digestion resistance and cell membrane permeability, 17 peptide sequences were comprehensively screened for molecular docking.

[0070] (4) Molecular docking virtual screening of oyster peptides:

[0071] The peptides screened in the previous process were used as ligands, and Keap1 and α-glucosidase were used as receptors. The action sites and interaction forces between oyster peptides and Keap1 and α-glucosidase were analyzed by molecular docking technology.

[0072] The three-dimensional structures of Keap1 (4L7B) and α-glucosidase (3A4A) were obtained from the PDB database (http: / / www.rcsb.org / ). The three-dimensional structures of the peptides were constructed using the Pymol program. Semi-flexible docking of the oyster peptide to Keap1 (4L7B) and α-glucosidase (3A4A) was performed using the Autdock software. The docking results were expressed as binding energy values, and the conformation with the lowest binding energy was selected as the optimal binding site.

[0073] Nine peptide sequences showed docking binding energies with Keap1 below -8.0 kcal / mol. Eight peptide sequences showed docking binding energies with α-glucosidase below -6.0 kcal / mol. Five sequences were selected for visualization analysis based on potential activity, safety, and bioaccessibility to further explore the molecular mechanisms of virtual prediction of interactions between Keap1 and α-glucosidase. The five peptide sequences selected were PYR, FKP, LPPR, NWP, and PRPP. The potential oyster peptide sequences and their properties are shown in Table 1. The binding energies of the peptides PYR, FKP, LPPR, NWP, and PRPP with Keap1 are shown in Table 2. The binding energies of the peptides PYR, FKP, LPPR, NWP, and PRPP with α-glucosidase are shown in Table 3.

[0074] Among them, the interactions between PYR, FKP, LPPR, NWP, and PRPP and Keap1 are as follows Figure 2-11 The interaction with α-glucosidase is shown in Figure 12-21 shown.

[0075] Table 1 Screened potential oyster polypeptide sequences and related properties

[0076]

[0077] Table 2 Docking results of peptides and keap1 molecules

[0078]

[0079] Table 3 Docking results of peptides and α-glucosidase molecules

[0080]

[0081] As can be seen from Tables 2 and 3, the peptides PYR, FKP, LPPR, NWP, and PRPP screened by bioinformatics tools and molecular docking all have potential antioxidant and α-glucosidase inhibitory activities, and their activities will be verified after further synthesis.

[0082] Example 2: Chemical synthesis of polypeptide sequences and activity verification

[0083] The chemical synthesis was commissioned to Sangon Biotech (Shanghai) Co., Ltd. The liquid phase test results of the synthetic peptides PYR, FKP, LPPR, NWP, and PRPP were as follows: Figure 22-26 The mass spectrometry results are shown as Figure 27-31 shown.

[0084] The above sequences were selected for synthesis, and the peptide activities were preliminarily verified by the DPPH, ABTS, and ORAC assays. The antioxidant activities of the synthesized peptides PYR, FKP, LPPR, NWP, and PRPP were verified by the DPPH, ABTS, and ORAC assays, respectively, using the same concentration of reduced glutathione (GSH) as a control.

[0085] (1) The ability of peptides PYR, FKP, LPPR, NWP, and PRPP to scavenge DPPH free radicals

[0086] The ability of oyster antioxidant peptides to scavenge DPPH free radicals was determined by referring to the national standard GB / T 39100-2020, the first method of peptide antioxidant activity determination, the DPPH method. The results are as follows Figure 32 As shown in the figure, the polypeptides PYR, FKP, LPPR, NWP, and PRPP all have a certain ability to scavenge DPPH free radicals, but their ability to scavenge DPPH free radicals is inferior to that of GSH at the same concentration.

[0087] (2) Ability of peptides PYR, FKP, LPPR, NWP, and PRPP to scavenge ABTS free radicals

[0088] The ability of oyster antioxidant peptides to scavenge ABTS free radicals was determined according to the second method of the national standard GB / T 39100-2020 Determination of Peptide Antioxidant Activity (ABTS method). The results are as follows: Figure 33 As shown in the figure, PYR and NWP have certain ability to scavenge ABTS free radicals, among which the ability of PYR to scavenge ABTS free radicals is slightly inferior to that of GSH at the same concentration.

[0089] (3) ORAC activity of peptides PYR, FKP, LPPR, NWP, and PRPP

[0090] Prepare 75 mmol / L pH 7.4 phosphate buffer, 40 mmol / L AAPH (2,2-azobisisobutylamidine dihydrochloride) solution, 117 nM FL (fluorescein sodium) dilution solution, and 20 mM Trolox (water-soluble vitamin E analog) stock solution respectively.

[0091] Pipette 120 μL of FL solution into an empty 96-well fluorescent plate. Add 20 μL of various concentrations of oyster peptides PYR, FKP, LPPR, NWP, PRPP, GSH, or Trolox solution and shake for 5 minutes. Incubate at 37°C for 15 minutes, then quickly add 60 μL of AAPH solution to initiate the reaction. Fluorescence values ​​were measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Fluorescence values ​​(Fn) were measured every 3 minutes during the reaction. Fluorescence decay curves for different test substance concentrations were plotted with the measurement time as the abscissa and the fluorescence value as the ordinate.

[0092] The measurement results are expressed as ORAC values, and the calculation formula is:

[0093] ORAC(μmol TE / mg)=[(AUC s -AUC +AAPH ) / (AUC T -AUC -APPH )]×M T / M s

[0094] Where AUC represents the area under the fluorescence curve of each group, M T and M s are the concentrations of Trolox and the sample solution to be tested (Trolox-μmol / mL, sample-mg / mL), respectively.

[0095] The fluorescence decay curves of oyster polypeptides PYR, FKP, LPPR, NWP, PRPP and GSH are as follows: Figure 34 As shown in the figure, it can be seen that the ORAC activity of PYR and NWP is significantly better than that of GSH. The ORAC values ​​of PYR, NWP and GSH are calculated to be 14.39±0.56 μmol TE / mg, 12.96±0.38 μmol TE / mg and 1.488±0.09 μmol TE / mg, respectively.

[0096] Note: The concentration of oyster peptides PYR, FKP, LPPR, NWP, PRPP and GSH was selected as 200 μg / mL.

[0097] The above sequences were selected and the α-glucosidase inhibitory activity of the synthetic polypeptides PYR, FKP, LPPR, NWP, and PRPP was verified by α-glucosidase inhibitory activity experiments, with the same concentration of acarbose as a control.

[0098] 100 μL of peptide solutions with different gradient concentrations and 20 μL of α-glucosidase solution (0.2 U, prepared in 0.1 mol / L, pH = 6.8 phosphate buffer) were added to the ELISA plate, mixed, and incubated at 37°C for 10 min. Then, 40 μL of 2.5 mmol / L PNPG (p-nitrophenyl-α-D-pyranoglucopyranoside) solution was added. After incubation at 37°C for 15 min, 50 μL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction. The absorbance at 405 nm (denoted as A) was measured. 样品 Buffer was used instead of sample as blank group. The formula for calculating α-glucosidase inhibitory activity is: α-glucosidase activity inhibition rate (%) = (A 空白 – A 样品 ) / A 空白] × 100.

[0099] The α-glucosidase inhibitory activity of synthetic peptides PYR, FKP, LPPR, NWP, and PRPP was tested. Figure 35 As shown in the results, all five synthetic peptides have certain α-glucosidase inhibitory activity, especially when the concentration reaches 2 mg / mL, the α-glucosidase inhibitory activity of PYR and NWP is better than that of acarbose.

[0100] The above experiments have once again demonstrated that the active peptides PYR and NWP of the present invention have strong antioxidant activity and α-glucosidase inhibitory activity, and can be used to prepare products with enhanced antioxidant and blood sugar-lowering functions.

[0101] The above description is only a preferred embodiment of the present invention, which is only used to explain the present invention and does not limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can be easily made by replacement or modification based on the technical content disclosed in this specification. All changes and improvements made on the principles of the present invention fall within the scope of protection of the present invention.

Claims

1. An application of oyster active peptide in the preparation of anti-oxidation and auxiliary blood sugar lowering products, characterized in that: The amino acid sequence of the oyster active peptide is PYR.

2. The use according to claim 1, characterized in that The product is an antioxidant or a nutraceutical.

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