Marine source bioactive peptide with blood sugar reducing effect, preparation and application

Abstract

The invention discloses a marine-derived bioactive peptide with a blood sugar reducing effect, a preparation and application, and belongs to the technical field of bioactive peptides. The amino acid sequence of the bioactive peptide is at least one of FYP, FPF and YAPFPK. Three bioactive peptides with the blood sugar reducing effect are separated and purified from oysters through enzymolysis, ultrafiltration, gel chromatography, liquid chromatography-mass spectrometry and bioinformatics technologies. According to the present invention, chemical synthesis is performed according to the identified peptide fragment sequence, the hypoglycemic activity of the synthesized single peptide fragment and the compounded peptide fragment is detected, and the bioactive peptides have good alpha-glucosidase inhibition activity, such that the bioactive peptides have good application prospects in the preparation of the products with the hypoglycemic effect, and can be widely used in the preparation of the products with the hypoglycemic effect. And a foundation is laid for high value-added utilization of oysters 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 marine source bioactive peptide with hypoglycemic efficacy, a preparation and application. BACKGROUND

[0002] With the continuous rise of global diabetes prevalence, developing safe and efficient natural hypoglycemic active ingredients has become an important research direction in the fields of biological medicine and functional food. As a key target enzyme for regulating postprandial blood glucose, alpha-glucosidase inhibitors can effectively reduce blood glucose peak by delaying carbohydrate hydrolysis. However, the existing chemical synthesis inhibitors generally have gastrointestinal adverse reactions and other problems, while natural alpha-glucosidase inhibitory peptides are attracting more and more attention due to their high safety and small side effects.

[0003] Oysters are a kind of marine shellfish resources rich in high-quality protein and low in fat, and the protein hydrolysate thereof contains a variety of physiologically active peptide segments, which show significant application potential in reducing blood glucose. However, the current preparation technology of oyster hypoglycemic peptides still faces the following key challenges: first, the enzyme hydrolysis process parameters are not fully optimized, the traditional enzyme hydrolysis method is low in efficiency, and new strengthening technologies such as ultrasonic assistance are not effectively integrated to improve the yield of active peptides; second, the protease screening system is not perfect, and it is difficult to accurately identify the most suitable enzyme for high-activity peptides; third, the separation and purification process needs to be optimized, and it is difficult to realize efficient enrichment of high-purity and high-activity target peptides; finally, the research on the structure-activity relationship and mechanism of action of active peptides is not deep enough, which to some extent limits the industrialization application process.

[0004] Therefore, developing marine source bioactive peptides with hypoglycemic efficacy from oysters not only can provide natural and safe hypoglycemic products, but also can promote the deep utilization and industrial development of oyster resources, which has important significance for promoting human health and economic development. SUMMARY

[0005] In view of the problems in the prior art, the purpose of the present application is to provide marine source bioactive peptides with hypoglycemic efficacy, a preparation and application.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows: The marine source bioactive peptide with hypoglycemic efficacy is characterized in that the amino acid sequence of the bioactive peptide is at least one of FYP, FPF and YAPFPK.

[0007] The application of the above-mentioned marine source bioactive peptide with hypoglycemic efficacy in preparing a product with hypoglycemic efficacy.

[0008] On the basis of the above-mentioned scheme, the product is food, medicine or health care product.

[0009] On the basis of the above scheme, the product further comprises food, pharmaceutical or health care acceptable adjuvant.

[0010] A preparation with blood sugar lowering efficacy, the effective component is at least one of FYP, FPF, YAPFPK.

[0011] On the basis of the above scheme, the concentration of the effective component is 2mg / mL.

[0012] On the basis of the above scheme, the blood sugar lowering is with ɑ-glucosidase inhibitory effect.

[0013] The advantages of the technical scheme of the present application are: The present application separates and purifies marine source bioactive peptides with blood sugar lowering efficacy from oysters through enzymolysis, ultrafiltration and gel chromatography, preliminarily identifies the components and sequences through liquid chromatography-mass spectrometry, further screens through bioinformatics technology, and finally obtains three bioactive peptides with better binding with receptor proteins through molecular docking analysis with ɑ-glucosidase. The identified peptide sequences are chemically synthesized, and the blood sugar lowering activity of the synthesized single peptide and compound peptide is detected. These bioactive peptides have better ɑ-glucosidase inhibitory activity, thus having better application prospect in the preparation of products with blood sugar lowering efficacy, laying a foundation for high value-added utilization of oysters and promoting the research and application of functional active peptides. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Inhibition rate of each component separated by ultrafiltration on ɑ-glucosidase; Figure 2 Chromatographic separation diagram obtained by Sephadex G-30 gel filtration; Figure 3 Inhibition rate of four components obtained by gel filtration on ɑ-glucosidase; Figure 4 LC-MS / MS total ion chromatogram of F3 component; Figure 5 Secondary structure diagram of 10 oyster active peptides screened; Figure 6 Interaction analysis diagram of oyster active peptides FYP (a), FPF (b) and YAPFPK (c) with ɑ-glucosidase; Figure 7 Tandem mass spectrum diagram of chemically synthesized FPF (a), FYP (b) and YAPFPK (c); Figure 8 Influence of different proteases under ultrasonic and non-ultrasonic conditions on hydrolysis degree; Figure 9 Effects of different proteases on the inhibition rate of α-glucosidase under ultrasonic conditions Figure 10 Effects of enzymolysis time on the degree of hydrolysis and the inhibition rate of α-glucosidase of oyster protein peptides Figure 11 Effects of enzyme amount on the degree of hydrolysis and the inhibition rate of α-glucosidase of oyster protein peptides Figure 12 Effects of substrate concentration on the degree of hydrolysis and the inhibition rate of α-glucosidase of oyster protein peptides Figure 13 Response surface plots of each experimental factor on the degree of hydrolysis (DH); Figure 14 Response surface plots of each experimental factor on the inhibition rate of α-glucosidase; Figure 15 Inhibition activities of FPF, FYP and YAPFPK on α-glucosidase Figure 16 Inhibition activities of FPF+FYP, FPF+YAPFPK and FYP+YAPFPK on α-glucosidase. DETAILED DESCRIPTION

[0015] The terms used in the present application have the meanings generally understood by those of ordinary skill in the art, unless otherwise defined. The present application is described in further detail below in conjunction with specific examples and with reference to the data. The following examples are merely intended to illustrate the present application and not to limit the scope of the present application in any way.

[0016] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The experimental materials, reagents, and medicines used in the following examples can be purchased through general channels, unless otherwise specified.

[0017] In the following examples, The method for measuring the degree of hydrolysis is as follows: During the enzymolysis of proteins, proton absorption and release always occur, and thus the pH of the system must change as the enzymolysis proceeds. In a neutral or slightly alkaline environment, the degree of protonation of amino groups is low, while the degree of dissociation of carboxyl groups is high. The release of protons leads to a decrease in the pH of the system, and thus the constant pH needs to be maintained by adding alkali, and the amount of alkali consumed is proportional to the number of peptide bonds broken. According to the amount of alkali consumed, the degree of hydrolysis of proteins can be calculated according to the following formula.

[0018] As the protein releases protons during hydrolysis, the pH value of the solution will drop sharply. At this time, 0.1M NaOH solution is added to maintain the pH. The degree of hydrolysis (DH) can be calculated by the volume of alkali solution added. The calculation formula is as follows: DH=(h / htot)×100%=[(C×V) / (a×m×htot)]×100% C, concentration of NaOH solution, moL / L; V, volume of NaOH solution consumed, mL; ɑ, dissociation degree of ɑ-amino group: ɑ=10pH-pKa / (1+10pH-pKa); Wherein, pH is the pH value of the hydrolysis system, pKa is the pKa of the amino group, pKa=7.8+[(298-T)×2400] / (298×T), where T is the Kelvin temperature; m, mass of oyster protein in solution, g; htot, the total number of peptide bonds in the substrate protein, mmol / g, for oyster protein hot = 8.3 (calculated based on the amino acid composition of oyster protein).

[0019] In vitro α-glucosidase activity inhibition rate determination: The experiment of oyster peptide inhibiting α-glucosidase activity was carried out using the method of Kim et al. with slight modifications.

[0020] α-glucosidase was prepared at a concentration of 0.2 U / mL using 0.1 M sodium phosphate buffer, pH 7.4, and p-nitrophenyl-α-D-glucoside (pNPG) was prepared at a concentration of 0.9 mg / mL using ultrapure water. 20 μL of sample peptide solution and 10 μL of α-glucosidase were added to the experimental groups, respectively. The mixture was then preheated in a 37°C incubator for 15 min. After removal, 40 μL of pNPG was immediately added to initiate the reaction. After mixing, the mixture was incubated in a 37°C incubator for 5 min. The reaction was terminated by the addition of 140 μL of 1 M Na2CO3 solution. The experiment also included blank, control, and sample background groups. The blank group added only 40 μL of the substrate pNPG, with the remainder replaced by equal volumes of buffer and water. This eliminated interference from the spontaneous conversion of pNPG to the chromogenic product 4-nitrophenol (PNP) during the preheating process. The control group was identical to the experimental group except that the sample peptide solution was omitted. The peptide solution was replaced by an equal volume of water. Under the same conditions, the maximum content of the chromogenic product PNP generated by the reaction between α-glucosidase and the substrate pNPG was determined. The sample background group added only the sample peptide solution, with the remainder replaced by an equal volume of buffer. This eliminated the influence of the absorbance of the sample peptide solution at the corresponding wavelength. After the reaction was terminated, the PNP content of the reactant was measured at a wavelength of 405 nm using a spectrophotometer and recorded. The reagent amounts used for each group are shown in Table 1.

[0021] The calculation formula of the inhibition rate of a-glucosidase activity is as follows: a-glucosidase activity inhibition rate (%) = (1-ODs-ODd / ODc-ODb) x 100 ODb: blank group absorbance value, absorbance value of the solution in which the peptide solution and a-glucosidase solution are replaced with an equal amount of buffer and water; ODc: control group absorbance value, absorbance value of the solution in which the peptide solution is replaced with an equal amount of water; ODs: experimental group absorbance value; ODd: sample background group absorbance value, absorbance value of the solution containing only oyster peptide solution and buffer.

[0022] Table 1: Reagent dosage of each group The source of oysters used in the following examples is the Qingdao Chengyang Aquatic Products Wholesale Market in China.

[0023] Example 1 A marine-derived bioactive peptide with hypoglycemic effect, the bioactive peptide being a peptide of at least one of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3: SEQ ID NO: 1: FYP; SEQ ID NO: 2: FPF; SEQ ID NO: 3: YAPFPK.

[0024] Example 2 A method for extracting a marine-derived bioactive peptide with hypoglycemic effect from oysters, the steps being as follows: (1) Shell the oysters, remove the meat and internal organs, and homogenize according to a material-to-liquid ratio of 30% (oyster meat: water = 10 g: 33.33 mL). After homogenization, perform ultrasonic treatment (450 W, 25 min), heat at 90°C for 10 min, add alkaline protease at a ratio of 3100 U / g (3100 U of enzyme per gram of substrate), and hydrolyze at the optimal temperature and pH (temperature 50°C, pH 10) of the alkaline protease for 3 h; maintain the pH of the system at the optimal pH by using 1 mol / L NaOH during the reaction process; after the enzymatic hydrolysis is complete, place the hydrolysis solution in a 100°C water bath to inactivate the enzyme, and maintain the temperature for 10 min; after cooling, centrifuge at 8000 r / min for 30 min at 4°C, take the supernatant, and freeze-dry to obtain oyster protein peptides.

[0025] (2) After enzymatic hydrolysis, the oyster protein solution was first filtered through a membrane, and then the oyster protein hydrolysis product was ultrafiltered using an ultrafiltration centrifuge tube to obtain enzymatic hydrolysates of >10 kDa, 3-10 kDa, and <3 kDa. The collected enzymatic hydrolysates were dried using a vacuum freeze-drying device to obtain freeze-dried powder. The inhibition rates of the components >10 kDa, 3-10 kDa, and <3 kDa on α-glucosidase were determined, and the results were as follows: Figure 1 shown.

[0026] Depend on Figure 1 The <3 kDa fraction exhibited a 52.02% inhibition rate against α-glucosidase, significantly higher than the other two groups. Most studies have also demonstrated that small-molecular-weight peptides exhibit stronger α-glucosidase inhibitory activity because they more readily interact with the active site of α-glucosidase, exerting their inhibitory effect. Therefore, the <3 kDa fraction was selected for further isolation and purification.

[0027] (3) The fraction <3 kDa was purified using a gel chromatography device. The diameter-to-height ratio of the Sephadex G-30 gel chromatography device column was set to 1.6:70. The sample was loaded at a concentration of 30 mg / mL and a volume of 1.0 mL. The column was eluted with deionized water (pH 10) and fractions were collected at a flow rate of 1 mL / min. Fractions were detected at 280 nm and collected in the order of peak elution time and freeze-dried in a vacuum. The α-glucosidase inhibition rate of each fraction was determined.

[0028] The chromatographic separation obtained by Sephadex G-30 gel filtration is shown in FIG. Figure 2 As shown, the absorbance of the separated solution was measured at an ultraviolet wavelength of 280 nm. The fraction <3 kDa was separated to obtain four peaks, and the absorbance remained stable thereafter. According to the gel column separation principle, the peptide with large molecular weight flows out of the column first, so the first peak to elute is F1, with the largest molecular weight, and the last peak to elute is F4, with the smallest molecular weight. The four fractions were collected to determine their effects on the α-glucosidase inhibitory activity. The results are shown in Figure 2. Figure 3 As shown, the α-glucosidase inhibition rates of the four fractions varied, reaching 30.38%, 35.99%, 52.10%, and 44.01%, respectively. Fraction F3 exhibited the highest α-glucosidase inhibition rate, significantly different from the other fractions. Therefore, repeated collections of F3 were performed to identify the peptide sequences within them using mass spectrometry.

[0029] (4) Amino acid sequence mass spectrometry identification The amino acid sequence of component F3 was determined by liquid chromatography-mass spectrometry. The liquid phase was EasynLC1200 nanoliter liquid phase system. The sample was desalted and reserved on the pre-column, and then separated by the analysis column. The analysis column was C18 reversed-phase chromatography (Acclaim Pep MapRSLC, 75 μm x 25 cm C18 2 μm 100A). The gradient used in the experiment was that the flow phase B (80% acetonitrile, 0.1% formic acid) was increased from 5% to 38% within 30 min. The mass spectrometry was performed by Thermo Fisher Q Exactive system (Thermo Fisher, USA) combined with nanospray NanoFlex ion source (Thermo Fisher, USA), with a spray voltage of 1.9 kV and an ion transmission tube heating temperature of 275 °C. The mass spectrometry scanning mode was data-dependent acquisition mode (DDA, Data Dependent Analysis), with a first mass spectrometry scanning resolution of 70000 and a scanning range of 350 2000 m / z, and a maximum injection time of 100 ms. A maximum of 20 secondary spectra with a charge of 2+ to 5+ were collected in each DDA cycle, and the maximum injection time of the secondary mass spectrometry ion was 50 ms. The collision chamber energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, which was suitable for all precursor ions, and the dynamic exclusion setting was 25 s. The pyropia database (2023 version 23, 24045) downloaded from UniProt was used for peak DB search, with a fragment ion mass tolerance of 0.02 Da and an expansion tolerance of 10 ppm.

[0030] The LC-MS / MS total ion chromatogram of the F3 component is shown in Figure 4 The raw data file of mass spectrometry acquisition was processed and analyzed by PEAKS Online 11 (Bioinformatics Solutions Inc. Waterloo, Canada) software. The database was uniprot-Magallana gigas database, and 2530 peptide segments were retrieved.

[0031] (5) The peptide sequences identified by mass spectrometry are respectively calculated for their activity scores by PeptideRanker (https: / / www.expasy.org / resources / peptidecutter) according to ① relative molecular mass (MW) ≤ 1000, ② no peptide segment modification; the peptide segments with a score > 0.5 are selected; the water solubility of the peptide segments is predicted by Innovagen; the toxicity of the peptide segments is predicted by ToxinPred3.0 website, and the peptide segments with a toxicity threshold prediction < 0.4 are selected; and the physicochemical properties of the active peptides such as hydrophobicity, hydrophilicity, isoelectric point pi and charge are analyzed by a proteomics website. The target peptide sequences of high biological activity, good water solubility and no toxicity are screened from the oyster proteins.

[0032] Ten peptide segments are finally screened, and the secondary structures of the 10 peptide segments are shown in Table 2. Figure 5

[0033] Table 2 shows the information of the oyster active peptides identified in Table 2. (6) Analysis of molecular docking interaction The 3D structure of the oyster peptide obtained by the above screening is generated by PepDraw to verify the effectiveness of the screened peptide and the receptor, and the receptor and the ligand are subjected to molecular docking. The protein receptor structure file is obtained in PDB format from the RCSB PDB database (http: / / www.rcsb.org / ), and the Autodock Tools 1.5.6 software is used to remove water molecules and hydrogenated proteins. The processed receptor and ligand files are imported into the Autodock Tools 1.5.6 software for molecular docking verification test. The size of the docking box is set to 30A x 30A x 30A, the active center of alpha-glucosidase (PDB ID: 3A4A) is as follows: x = 16.987, y = -8.928, z = -15.381, and finally the PyMol software is used to construct a three-dimensional schematic diagram. The data used for simulation include the protein receptor file, the ligand (.pdbqt) file and the grid size.

[0034] The binding energy represents the spontaneous degree of binding between the receptor and the ligand, and the smaller the value, the stronger the affinity and the higher the degree of binding. The binding pocket and center coordinates of the 10 oyster polypeptides and the 3A4A protein crystal are shown in Table 3.

[0035] Table 3 shows the binding pocket and center coordinates of the peptide and the 3A4A protein crystal. ​Docking: The binding energy absolute values ​​of the 10 peptides were ranked from highest to lowest. The results are shown in Table 4. Larger absolute values ​​of docking energy indicate better binding between the peptide and the α-glucosidase receptor protein. Therefore, it is assumed that the peptides FYP (-6.71 kcal / mol), FPF (-6.16 kcal / mol), and YAPFPK (-4.01 kcal / mol) may possess significant α-glucosidase inhibitory activity and are intended for further chemical synthesis and activity verification.

[0036] Table 4 Docking energy of peptides and α-glucosidase molecules Among them, the interaction analysis of peptides FYP, FPF and YAPFPK with α-glucosidase (PDB ID: 3A4A) is as follows Figure 6 shown.

[0037] (7) Three peptides, FYP, FPF and YAPFPK, were synthesized by solid phase synthesis. The mass spectra of the three peptides are shown in Figure 2. Figure 7 As shown, a desalted peptide with a purity greater than 98% was obtained, namely a bioactive peptide with hypoglycemic effect.

[0038] Example 3 Effects of protease types and ultrasonic treatment on the hydrolysis degree and α-glucosidase activity inhibition of oyster protein peptides Eight equal portions of oyster meat homogenate were weighed and homogenized at a 30% material-to-liquid ratio (oyster meat: water = 10 g: 33.33 mL). After homogenization, four groups were sonicated (450 W, 25 min), while the other four groups were not. Four proteases (neutral protease, alkaline protease, papain, and trypsin) were added to the sonicated and unsonicated groups, respectively, and mixed thoroughly. The protease dosage was 3000 U / g (3000 U per gram of substrate). Hydrolysis was performed for 3 h at the optimal temperature and pH for each protease. The pH of the system was maintained constant with 1 mol / L NaOH throughout the reaction (the optimal pH was maintained throughout). The optimal temperature and pH for each protease are shown in Table 5. After hydrolysis, the samples were inactivated in a 100°C water bath for 10 min. The samples were cooled to room temperature, adjusted to pH 7.0, and centrifuged at 8000 rpm for 30 min at 4°C. The supernatant was collected. The amount of NaOH consumed during the 3-hour hydrolysis was recorded, and the degree of hydrolysis was used as an evaluation index to evaluate the enzymatic hydrolysis effect with and without ultrasound. The inhibitory rate of the supernatant after hydrolysis on α-glucosidase activity was determined to determine the optimal protease.

[0039] Table 5 Optimum temperature and pH of different proteases Effect of ultrasonic treatment on degree of hydrolysis of oyster protein peptides Figure 8 As shown in Table 2, compared with the non-ultrasonic treatment group, the degree of hydrolysis of each enzyme was increased after ultrasonic treatment (480 W, 25 min), and the degree of hydrolysis of neutral protease, papain, alkaline protease and trypsin reached 11.12%, 5.28%, 18.12% and 9.71%, respectively, which was increased by 2.95%, 2.01%, 3.97% and 2.34%, respectively, compared with the non-ultrasonic treatment group. Thus, ultrasonic treatment can improve the degree of hydrolysis of oyster protein peptides.

[0040] Effect of the type of protease on the α-glucosidase inhibitory activity of oyster protein peptides under ultrasonic assistance Figure 9 As shown in Table 3, the α-glucosidase inhibitory activity of oyster protein peptides obtained by using four proteases was arranged from strong to weak as follows: alkaline protease > neutral protease > trypsin > papain. Among them, the inhibitory activity of peptides obtained by alkaline protease was the best, and the α-glucosidase inhibitory rate was 50.36%.

[0041] Example 4 Effect of enzyme hydrolysis conditions on the degree of hydrolysis of oyster protein peptides and α-glucosidase activity inhibition (1) Effect of enzyme hydrolysis time on the degree of hydrolysis of oyster protein peptides and α-glucosidase activity inhibition The oysters were shelled and the meat was removed from the viscera, and then homogenized at a ratio of 30% (oyster meat: water = 10 g: 33.33 mL). After homogenization, ultrasonic treatment (450 W, 25 min) was performed, and then heated at 90°C for 10 min. Alkaline protease was added at a ratio of 3000 U / g (3000 U per gram of substrate), and then hydrolyzed at the optimal temperature and pH (temperature 50°C, pH 10) for 1 h, 2 h, 3 h, 4 h, 5 h, respectively. During the reaction, 1 mol / L NaOH was used to maintain the pH of the system (always maintained at the optimal pH). After the completion of enzyme hydrolysis, the enzyme hydrolysis solution was placed in a water bath at 100°C for enzyme inactivation for 10 min. After cooling, centrifugation was performed at 8000 r / min for 30 min at 4°C, and then the supernatant was taken and freeze-dried to obtain oyster protein peptides.

[0042] The amount of NaOH consumed during the hydrolysis process was recorded, and the degree of hydrolysis was used as an evaluation index to evaluate the effect of enzyme hydrolysis time on the degree of hydrolysis of oyster protein peptides. The inhibitory rate of the supernatant after hydrolysis on α-glucosidase activity was determined to determine the optimal enzyme hydrolysis time. The results are shown in Table 6 and Figure 10 .

[0043] Table 6 Effect of enzyme hydrolysis time on the degree of hydrolysis of oyster protein peptides and α-glucosidase activity inhibition From Table 6 and Figure 10 It can be seen that: with the increase of enzymolysis time, the degree of hydrolysis shows an upward trend, and the degree of hydrolysis and the inhibition rate of α-glucosidase increase within the first 3h of the hydrolysis reaction, which is due to the continuous cutting of the substrate protein into small molecular polypeptides with α-glucosidase inhibitory activity by protease, and such polypeptide fragments continue to increase; when the hydrolysis time reaches 3h, the inhibition rate reaches the highest, and then decreases with time, while the degree of hydrolysis continues to rise but has no significant difference. The reason for this situation should be that the effective peptide segments with α-glucosidase inhibitory effect are continuously hydrolyzed into small peptides without α-glucosidase inhibitory activity, so the optimal enzymolysis time is 3h.

[0044] (2) Effect of enzyme addition amount on degree of hydrolysis of oyster protein peptide and inhibition of α-glucosidase activity The oysters were shelled and the meat was removed from the viscera, and homogenized according to a material liquid ratio of 30% (oyster meat: water = 10 g: 33.33 mL). After homogenization, ultrasonic treatment (450 W, 25 min), heating at 90℃ for 10 min, alkaline protease was added at a ratio of 1000U / g, 2000U / g, 3000U / g, 4000U / g, 5000U / g respectively, and hydrolysis was carried out at the optimal temperature and pH (temperature 50℃, pH 10) of alkaline protease for 3h; during the reaction, 1 mol / L NaOH was used to maintain the pH of the system (always maintained at the optimal pH); after the enzymolysis was completed, the enzymolysis solution was placed in a 100℃ water bath for enzyme inactivation operation, and the time was 10 min; after cooling, centrifugation at 8000 r / min for 30 min at 4℃, the supernatant was taken, and freeze-drying was carried out to obtain oyster protein peptide.

[0045] The amount of NaOH consumed during the hydrolysis process was recorded, and the degree of hydrolysis was used as an evaluation index to evaluate the effect of enzyme addition amount on the degree of hydrolysis of oyster protein peptide. The inhibition rate of the supernatant after hydrolysis on α-glucosidase activity was determined to determine the optimal enzyme addition amount. The results are shown in Table 7 and Figure 11

[0046] Table 7 Effect of enzyme addition amount on degree of hydrolysis of oyster protein peptide and inhibition of α-glucosidase activity From Table 7 and Figure 11 ​It can be seen that as the enzyme dosage increases, the degree of hydrolysis gradually increases because more enzymes are available for substrate hydrolysis. When the enzyme dosage reaches 3000 U / g, the degree of hydrolysis gradually tends to be flat. However, when the enzyme dosage is too high, the hydrolysis reaction may proceed excessively, resulting in the generated product no longer having an inhibitory effect but becoming a free amino acid. This excessive hydrolysis may be due to the saturation of enzyme-substrate interaction, and even increasing the amount of enzyme cannot increase the amount of hydrolysis products. In addition, excessive enzyme dosage may cause competition between enzymes, making the hydrolysis products more prone to further degradation into free amino acids rather than maintaining their original inhibitory activity. Therefore, the optimal amount of enzyme is 3000 U / g.

[0047] (3) Effect of substrate concentration on the degree of hydrolysis of oyster protein peptides and the inhibition of α-glucosidase activity The oysters were shelled and the meat was removed from the viscera, and then homogenized at a ratio of 10%, 20%, 30%, 40%, and 50% of the substrate. After homogenization, ultrasonic treatment was performed (450 W, 25 min), heating at 90°C for 10 min, and then adding alkaline proteinase at a ratio of 3000 U / g. The hydrolysis was carried out at the optimal temperature and pH of alkaline proteinase (temperature 50°C, pH 10) for 3 h. During the reaction, 1 mol / L NaOH was used to maintain the pH of the system (always maintained at the optimal pH). After the enzymatic hydrolysis was completed, the enzyme solution was placed in a water bath at 100°C for 10 min to inactivate the enzyme. After cooling, centrifugation was performed at 8000 r / min for 30 min at 4°C, and the supernatant was taken and freeze-dried to obtain oyster protein peptides.

[0048] The amount of NaOH consumed during the hydrolysis process was recorded, and the degree of hydrolysis was used as an evaluation index to evaluate the effect of substrate concentration on the degree of hydrolysis of oyster protein peptides. The inhibition rate of the supernatant after hydrolysis on the activity of α-glucosidase was determined to determine the optimal substrate concentration. The results are shown in Table 8 and Figure 12

[0049] Table 8 Effect of substrate concentration on the degree of hydrolysis of oyster protein peptides and the inhibition of α-glucosidase activity According to Table 8 and Figure 12 It can be seen that when the substrate concentration is 30%, the α-glucosidase inhibitory activity is the highest (55.58%), and then it rapidly decreases with increasing concentration; the degree of hydrolysis reaches a peak value (18.95%) at 30% and then decreases slightly. At low concentrations, alkaline protease can fully hydrolyze the substrate to generate highly active peptide segments, and the inhibition rate and degree of hydrolysis increase with increasing concentration; but when the concentration exceeds the optimal concentration, the viscosity of the system is too large to limit the efficiency of enzymatic hydrolysis. Therefore, the optimal substrate concentration is 30%.

[0050] Example 5 ​Response surface experiment was used to optimize the enzymatic hydrolysis conditions The hydrolysis time (A), enzyme dosage (B), and substrate concentration (C) were selected as the factors for investigation, and the response surface experiment was carried out with three levels for each factor. The results are shown in Table 9.

[0051] Table 9: Response surface experiment data The α-glucosidase inhibitory activity of oyster peptide was subjected to quadratic polynomial regression fitting, and the quadratic regression equation of the inhibition rate and the hydrolysis time, enzyme dosage, and substrate concentration was obtained as follows: The fitting equation of the hydrolysis degree (%) = -57.7662 + 0.0170795 * A + 1.59657 * B + 17.2832 * C - 1.65e-05 * AB + 3.5 * AC - 0.01325 * BC - 2.70825 * A 2 -0.0252075 * B 2 -2.73575 * C 2 The fitting equation of the α-glucosidase inhibitory rate (%) = -72.0262 + 0.0397285 * A + 3.16685 * B + 12.2885 * C + 0.00015675 * AB - 4.75 * AC + 0.00075 * BC - 7.23 * A 2 -0.0603725 * B 2 -1.96725 * C 2 The regression model of the hydrolysis degree of oyster peptide was subjected to variance analysis, as shown in Table 10. The results showed that the model was highly significant (P<0.0001), and the non-significant term was not significant (P>0.05), indicating that the regression equation had a high fitting degree for the experimental results, and thus the regression equation could be used to analyze and predict the actual values. The standard for the fitting degree between the predicted value and the actual value was that the model determination coefficient value had to be greater than 0.8, and the fitting degree R 2 of the model was 0.9855, i.e., 98.55% of the change in the response value of the hydrolysis degree was derived from the selected variables, indicating that the model could well predict the hydrolysis degree of oyster peptide. The coefficient of variation (CV) was an index indicating whether the model was reliable and accurate, and the smaller the value, the better the model. The model variation coefficient in this experiment was 3.03%, indicating that the model had high reliability. In addition, the signal-to-noise ratio (AP) of the model was 17.9430>4, indicating that the fitting degree of the model was good.

[0052] Table 10 Analysis of variance table of DH In linear effect, the single factor enzyme amount (A) had significant effect on the degree of hydrolysis (P<0.05), but the effect of substrate concentration (B) on the response value was not significant (P>0.05), and the effect of single factor enzymolysis time (C) on the degree of hydrolysis was not significant (P>0.05); in the surface effect, factor C 2 had significant effect on the degree of hydrolysis, and factors A 2 and B 2 had significant effect on the degree of hydrolysis. It can be seen from Figure 13 that the contour plot of factors enzyme amount (A) and substrate concentration (B) was elliptical, indicating that the interaction of AB had high significant effect on the degree of hydrolysis of oyster peptide; analysis of these results showed that the degree of hydrolysis of oyster peptide was not a simple linear relationship with each factor, and the quadratic term and interaction of some factors also affected the change of response value to a great extent.

[0053] The regression model of α-glucosidase inhibitory rate of oyster peptide was subjected to analysis of variance, as shown in Table 11. The results showed that the model was highly significant (P<0.001), and the lack of fit was not significant (P>0.05), indicating that the fitting degree of the regression equation to the experimental results was high, and therefore the regression equation could be used to analyze and predict the actual value of the experiment. The standard of high fitting degree between the predicted value and the actual value of the model was that the model determination coefficient value had to be greater than 0.8, and the fitting degree R 2 of the model was 0.9866, that is, 98.66% of the change of response value α-glucosidase inhibitory rate was derived from the selected variables, indicating that the model could well predict the enzyme inhibitory activity of oyster peptide. The coefficient of variation (CV) was an index indicating whether the model was reliable and accurate, and the smaller the value represented the better the model. The model variation coefficient of the experiment was 1.91%, indicating that the reliability of the model was high. In addition, the signal-to-noise ratio (AP) of the model was 21.8155>4, indicating that the fitting degree of the model was good.

[0054] Table 11 Analysis of variance table of α-glucosidase activity inhibitory rate In linear effect, the single factor enzyme amount (A), substrate concentration (B), and enzymolysis time (C) had no significant effect on the response value (P>0.05); in the surface effect, factors A 2 , B 2 and C 2 had significant effect on the α-glucosidase inhibitory rate. It can be seen from Figure 14It can be seen that the contour plot of factor enzyme amount (A) and substrate concentration (B) is elliptical, indicating that the interaction of AB is highly significant to the inhibitory activity of oyster peptide on α-glucosidase; analysis of these results shows that the inhibitory effect of oyster peptide on α-glucosidase activity is not a simple linear relationship with each factor, and the quadratic term of some factors and the interaction greatly affect the change of response value.

[0055] The regression model analysis is performed by Desgin-Expert 13 software, and the optimal process conditions of oyster enzymolysis are obtained as follows: enzyme amount 3055.18 U / g, solid-liquid ratio 30 %, enzymolysis time 3.09031 h, and degree of hydrolysis 19.2199 %. Under the conditions, the α-glucosidase inhibitory rate of the enzymolysis product is 55.551 %. Starting from the actual operation, the process conditions are adjusted as follows: enzyme amount 3100 U / g, solid-liquid ratio 30 %, and enzymolysis time 3 h. After the test verification under the conditions, the degree of hydrolysis of the enzymolysis product is 19.95 %, and the α-glucosidase inhibitory rate is 56.52 %. The result is very close to the predicted value, indicating that the model can well reflect the enzymolysis conditions of oyster hypoglycemic peptide.

[0056] Example 5 A marine source bioactive peptide preparation with hypoglycemic efficacy, the effective component being at least one of the bioactive peptides of FYP, FPF and YAPFPK.

[0057] FYP, FPF and YAPFPK are prepared into solutions with a concentration of 2 mg / mL by using ultrapure water, and the α-glucosidase inhibitory rates of the three are determined, and the results are shown in Table 1. Figure 15 The α-glucosidase inhibitory rates of the three peptides are FYP>FPF>YAPFPK. This is because the amino acid composition and conformation of FYP are more suitable for the enzyme active center, and the binding force is strong; the YAPFPK peptide chain is long, and the steric hindrance is large, and the binding efficiency with the enzyme active center is low.

[0058] The three peptides are compounded in pairs according to a mass ratio of 1:1, and are prepared into solutions with a concentration of 2 mg / mL by using ultrapure water; the concentration of each peptide in the solution is 2 mg / mL, and the α-glucosidase inhibitory rates of the two combinations are determined, and the results are shown in Table 2. Figure 16 With the change of peptide combination, the α-glucosidase inhibitory rates show a significant difference of FPF+FYP>FYP+YAPFPK>FPF+YAPFPK, and FPF+FYP are both short peptides, which combine different active pockets / auxiliary sites of α-glucosidase, and enhance the inhibition through “multi-target synergistic blocking”.

[0059] The above merely describes preferred embodiments of the present application, but is not intended to limit the present application to other forms, and any person skilled in the art can make changes or modifications to the above disclosed technical contents into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments without departing from the technical solution content of the present application and according to the technical essence of the present application still belongs to the protection scope of the technical solution of the present application.

Claims

1. Marine-derived bioactive peptide having hypoglycemic efficacy, characterized in that, The amino acid sequence of the bioactive peptide is at least one of FYP, FPF, and YAPFPK.

2. Use of the bioactive peptide from marine sources with hypoglycemic effect in claim 1 in the preparation of a product with hypoglycemic effect.

3. The use of the marine-derived bioactive peptide having blood glucose-lowering efficacy according to claim 2 in the preparation of a product having blood glucose-lowering efficacy, characterized in that, The product is food, medicine or health care product.

4. The use of the marine-derived bioactive peptide having blood glucose-lowering efficacy according to claim 3 in the preparation of a product having blood glucose-lowering efficacy, characterized in that, The product further comprises food, pharmaceutical or health care product acceptable adjuvants.

5. A preparation having a blood sugar-lowering effect, characterized by, The effective component is at least one of FYP, FPF and YAPFPK.

6. The preparation having blood sugar-lowering efficacy according to claim 5, wherein the preparation is a composition of a mixture of the extract of the plant of the genus Asparagus and the extract of the plant of the genus Lycium. The concentration of the effective component is 2 mg / mL.

7. The preparation having blood sugar lowering efficacy according to claim 5 or 6, wherein the preparation is a food or a food additive. The hypoglycemic effect is ɑ-glucosidase inhibition.

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