Small molecule peptide FDRIW with antioxidant activity and application thereof

Abstract

The invention discloses a small molecule peptide FDRIW with antioxidant activity and application thereof, and belongs to the technical field of enzymolysis processing products. The amino acid sequence of the small molecule peptide FDRIW is as shown in SEQ ID NO. 1. The small molecule peptide FDRIW is applied to preparation of a health care product with an antioxidant activity effect, and is applied to preparation of a medicine with an oxidative stress inhibition effect. The small molecule peptide with high scavenging activity on DPPH free radicals is obtained through screening, IC50 is equal to 3.63 mg / ml, and the small molecule peptide has the potential of being used as an antioxidant functional product.

Description

[0001] The present invention is a divisional application of CN 116064708 A. The application number of the original application is 202211204109.8, the application date is September 29, 2022, and the name of the invention is: Royal jelly polypeptides and small molecule peptides with antioxidant activity and their applications. Technical Field

[0002] The invention relates to a small molecule peptide FDRIW with antioxidant activity and application thereof, belonging to the technical field of enzymatic processing products. Background Art

[0003] Royal jelly is secreted by the pharyngeal and mandibular glands of young worker bees. It is composed of water, protein, carbohydrates, lipids, minerals and vitamins. It has high nutritional value and is widely used in food, biology and medical fields. It has multiple physiological functions such as anti-inflammatory, antibacterial, anti-tumor, and enhancing immunity. However, its processing and utilization level is very low, and its economic added value is low.

[0004] Bioactive peptides are functional peptide fragments produced during the hydrolysis, cleavage or maturation of proteins, and their relative molecular mass is usually less than 6000 Da. Common bioactive peptides include soybean peptides, glutathione, casein phosphopeptides, etc. Enzyme biotechnology is an efficient solution for the production of bioactive peptides and has been widely used in the production of bioactive peptides.

[0005] Oxidative stress is the cause of atherosclerosis, cardiovascular disease, cancer, diabetes and neurodegenerative diseases. The natural antioxidant system in the body often cannot meet the needs of the human body under external stimulation, and it is necessary to supplement antioxidants through diet or drugs to reduce oxidative stress in order to delay aging, improve sub-health status and reduce the incidence of diseases. Synthetic antioxidants such as BHA, BHT, gallic acid, etc. have good antioxidant effects, but these substances have certain side effects. Natural and safe antioxidant substances are a hot topic in current research. Summary of the invention

[0006] In view of the above-mentioned prior art, the present invention provides a small molecule peptide FDRIW with antioxidant activity and application thereof.

[0007] The present invention is achieved through the following technical solutions: A small molecule peptide FDRIW with antioxidant activity, whose amino acid sequence is shown in SEQ ID NO.1.

[0008] The small molecule peptide has good DPPH free radical scavenging activity, can be used to prepare health products with antioxidant activity, and can be used to prepare drugs with oxidative stress inhibition effect.

[0009] The present invention treats royal jelly by a specific method (enzymatic hydrolysis + gel filtration chromatography) to obtain royal jelly polypeptides with antioxidant activity. After LC-MS / MS identification, three small molecule peptides with high scavenging activity against DPPH free radicals were screened, namely: FDRIW (IC 50 =3.63 mg / ml), YPDWSW (IC 50 =3.59 mg / ml), WHDKIF (IC 50 =1.00 mg / ml), these three small molecule peptides all have the potential to be used as antioxidant functional products.

[0010] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A : Schematic diagram of the effect of protease on DPPH radical scavenging rate.

[0012] Figure 1B : Schematic diagram of the effect of protease on OH radical scavenging rate.

[0013] Figure 2A : Schematic diagram of the effect of enzymatic hydrolysis time on DPPH free radical scavenging rate.

[0014] Figure 2B : Schematic diagram of the effect of enzymatic hydrolysis time on OH radical scavenging rate.

[0015] Figure 3A : Schematic diagram of the effect of temperature on DPPH radical scavenging rate.

[0016] Figure 3B : Schematic diagram of the effect of temperature on OH radical scavenging rate.

[0017] Figure 4A : Schematic diagram of the effect of enzyme addition amount on DPPH free radical scavenging rate.

[0018] Figure 4B : Schematic diagram of the effect of enzyme addition amount on OH radical scavenging rate.

[0019] Figure 5A : Schematic diagram of the effect of solid-liquid ratio on DPPH radical scavenging rate.

[0020] Figure 5B : Schematic diagram of the effect of solid-liquid ratio on OH radical scavenging rate.

[0021] Fig. 6A : Schematic diagram of the effect of initial pH value on DPPH radical scavenging rate.

[0022] Figure 6B: Schematic diagram of the effect of initial pH value on OH radical scavenging rate.

[0023] Figure 7 :Schematic diagram of the effect of 1.5 mL / min flow rate on the separation effect of Sephadex G-15.

[0024] Figure 8 :Schematic diagram of the effect of 3.5 mL / min flow rate on the separation effect of Sephadex G-15.

[0025] Fig. 9 :Schematic diagram of the effect of 5.5 mL / min flow rate on the separation effect of Sephadex G-15.

[0026] Fig.10 :Schematic diagram of the effect of 7.5 mL / min flow rate on the separation effect of Sephadex G-15.

[0027] Fig.11 :Schematic diagram of the effect of 0.5 mL sample volume on the separation effect of Sephadex G-15.

[0028] Fig.12 :Schematic diagram of the effect of 1 mL sample volume on the separation effect of Sephadex G-15.

[0029] Fig.13 :Schematic diagram of the effect of 1.5 mL sample volume on the separation effect of Sephadex G-15.

[0030] Fig.14 :Schematic diagram of the optimal elution curve of Sephadex G-15.

[0031] Fig.15A : Schematic diagram of the results of determination of DPPH free radical scavenging rate of each component.

[0032] Fig. 15B : Schematic diagram of the results of the OH radical scavenging rate determination of each component.

[0033] Fig.16 : Basepeak diagram of sample mass spectrum.

[0034] Fig.17 : Mass spectrum of FDRIW.

[0035] Fig.18 : Mass spectrum of YPDWSW.

[0036] Fig.19 : Mass spectrum of WHDKIF. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0038] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0039] The royal jelly used in the following examples was purchased from Shandong Fengcai Health Industry Co., Ltd., placed in a sealed light-proof bag, and kept at -20°C for future use.

[0040] Example 1 Single factor optimization of royal jelly enzymatic hydrolysis process 1. Screening enzyme Papain, bromelain, neutral protease, alkaline protease and acid protease were selected for enzymatic hydrolysis of the raw materials. 2 g of pre-frozen royal jelly was accurately weighed, and the substrate solution was prepared at a material-liquid ratio of 1:8. Five proteases were added at an enzyme amount of 4000 U / g (enzyme activity / royal jelly). After reacting at 50℃ and 200 rpm for 4 h, samples were taken, enzymes were inactivated, cooled, centrifuged (8000rpm, 10 min), and supernatants were taken to determine the DPPH free radical scavenging rate and OH free radical scavenging rate.

[0041] Determination of DPPH free radical scavenging rate: Preheat the spectrophotometer / microplate reader for 30 min and adjust the wavelength to 517 nm. Take 500 μl of enzymatic hydrolysate, mix with 1000 μl of 0.2 mM DPPH solution (dissolved in anhydrous ethanol), shake well, react in a dark environment at room temperature for 20 min, and filter. Determine its absorbance Ai at 517 nm, replace the DPPH solution with 500 μl of anhydrous ethanol to measure the absorbance Aj, and replace the enzymatic hydrolysate with 500 μl of water to measure the absorbance A0. The formula for calculating the DPPH free radical scavenging rate is: DPPH scavenging rate = (1-Ai-Aj / A0) × 100%. BHA (butylated hydroxyanisole) was used as a control.

[0042] Determination of OH radical scavenging rate: Take 0.1 mL of the test solution, then accurately add 0.5 mL of salicylic acid-ethanol solution (9 mmol / L), 0.5 mL of ferrous sulfate solution (9 mmol / L) and 0.5 mL of H2O2 solution (8.8 mmol / L), mix well and react at 37°C in the dark for 30 min, and measure its absorbance Ai at 510 nm. At the same time, replace 0.5 mL of H2O2 solution with 0.5 mL of distilled water and measure its absorbance Aj; replace 0.1 mL of sample solution with 0.1 mL of distilled water and measure its absorbance A0. The formula for calculating OH radical scavenging rate is: OH radical scavenging rate = (1-Ai-Aj / A0) × 100%. Vc (vitamin C) is used as the control.

[0043] In this experiment, five proteases were selected to hydrolyze royal jelly. Figure 1A , Figure 1B As shown. Papain, bromelain, neutral protease, alkaline protease and acidic protease were used to enzymolyze royal jelly. In the DPPH free radical scavenging rate experiment, acidic protease had the best effect, followed by alkaline protease and neutral protease. In the OH free radical scavenging rate experiment, acidic protease, alkaline protease and neutral protease all had good free radical scavenging effects. Considering the protease effect and the added cost and other factors, acidic protease was selected as the enzyme preparation for further optimization.

[0044] 2. Optimization of enzymatic hydrolysis time Accurately weigh 2 g of pre-frozen royal jelly, prepare the substrate solution with a solid-liquid ratio of 1:8, add acidic protease at an enzyme dosage of 4000 U / g, adjust the initial pH value of the substrate solution to 4, react at 50 ℃ and 200 rpm for 3 h, 4 h, 5 h, 6 h, and 7 h, respectively, take samples, inactivate the enzyme, cool, and centrifuge (8000 rpm, 10 min), take a certain amount of supernatant to determine the DPPH free radical scavenging rate and OH free radical scavenging rate, and the determination method is the same as above.

[0045] The results are as follows Figure 2A , Figure 2B As shown in the figure. The DPPH free radical scavenging activity increased with the increase of enzymatic hydrolysis time in a short time. There was a significant difference between enzymatic hydrolysis for 3 or 4 hours and enzymatic hydrolysis for 5 hours (P<0.05), and there was no significant difference between enzymatic hydrolysis for 5 to 8 hours (P>0.05). The OH free radical scavenging activity was the highest when the enzymatic hydrolysis time was 5 hours, which was significantly different from other groups (P<0.05). Considering the enzymatic hydrolysis effect and enzymatic hydrolysis efficiency, 5 hours was finally selected as the enzymatic hydrolysis treatment time.

[0046] 3. Optimization of enzymatic hydrolysis temperature Accurately weigh 2 g of pre-frozen royal jelly, prepare the substrate solution with a solid-liquid ratio of 1:8, and the initial pH value is 4. Add acidic protease at an enzyme dosage of 4000 U / g. React at 30 ℃, 40 ℃, 50 ℃, 60 ℃, and 70 ℃ at 200 rpm for 4 h, then take samples, inactivate the enzyme, cool, and centrifuge (8000 rpm, 10 min) to take the supernatant, and determine the DPPH free radical scavenging rate and OH free radical scavenging rate using the same method as above.

[0047] The results are as follows Figure 3A , Figure 3B As shown. With the increase of enzymatic hydrolysis temperature, the DPPH free radical scavenging activity showed a trend of first increasing and then decreasing. Among them, when the enzymatic hydrolysis temperature was 50℃, the DPPH free radical scavenging activity was the highest, which had no significant difference with 60℃ (P>0.05), but was significantly higher than other groups (P<0.05). The OH free radical scavenging activity did not change much with temperature. Considering the enzymatic hydrolysis effect comprehensively, 50℃ was selected as the enzymatic hydrolysis treatment temperature.

[0048] 4. Optimization of enzyme dosage Accurately weigh 2 g of pre-frozen royal jelly, prepare the substrate solution with a solid-liquid ratio of 1:8, add acid protease at enzyme amounts of 2000, 4000, 6000, 8000, and 10000 U / g, react at 50℃ and 200 rpm for 5 h, take samples, inactivate the enzyme, cool, centrifuge (8000 rpm, 10 min), take the supernatant, and determine the DPPH free radical scavenging rate and OH free radical scavenging rate using the same method as above.

[0049] The results are as follows Figure 4A , Figure 4B As shown. With the increase of enzyme dosage, the DPPH radical scavenging rate of the enzymatic solution showed a gradual upward trend. There was no significant difference between the enzyme dosage of 6000 U / g and the enzyme dosage of 8000 U / g (P>0.05); there was a significant difference between the enzyme dosage of 8000 U / g and the enzyme dosage of 10000 U / g (P<0.05). The OH radical scavenging rate of the enzymatic solution had little relationship with the change of enzyme dosage. Considering the comprehensive cost, the enzyme dosage of 8000 U / g was selected for further experiments.

[0050] 5. Optimization of material-liquid ratio Accurately weigh 2 g of pre-frozen royal jelly and prepare substrate solutions with a material-liquid ratio of 1:3, 1:5, 1:7, 1:9, and 1:11, respectively. Add acidic protease at an enzyme dosage of 8000 U / g. After reacting at 50°C and 200 rpm for 5 h, take a sample, inactivate the enzyme, cool, and centrifuge (8000 rpm, 10 min) to take the supernatant, and determine the DPPH free radical scavenging rate and OH free radical scavenging rate using the same method as above.

[0051] The results are as follows Figure 5A , Figure 5B As shown. With the increase of the material-liquid ratio, the DPPH radical scavenging rate and OH radical scavenging rate of the enzymatic hydrolysate showed a gradually decreasing trend. There was no significant difference in the DPPH radical scavenging rate of the enzymatic hydrolysate between the material-liquid ratio of 1:5 and the material-liquid ratio of 1:3 (P>0.05). There was a significant difference in the OH radical scavenging rate of the enzymatic hydrolysate between the material-liquid ratio of 1:5 and the material-liquid ratio of 1:3 (P<0.05). Taking all factors into consideration, the material-liquid ratio of 1:3 was selected for the next experiment.

[0052] 6. Initial pH Optimization Accurately weigh 2 g of pre-frozen royal jelly, prepare the substrate solution with a solid-liquid ratio of 1:3, adjust the initial pH of the substrate solution to 2, 3, 4, 5, or 6 with 1M hydrochloric acid or 1M NaOH solution, add acid protease at an enzyme dosage of 8000 U / g, react at 50°C and 200 rpm for 5 h, take a sample, inactivate the enzyme, cool, and centrifuge (8000 rpm, 10 min), take the supernatant to determine the DPPH free radical scavenging rate and OH free radical scavenging rate, using the same determination method as above.

[0053] The results are as follows Fig. 6A , Figure 6B As shown. With the increase of initial pH value, the DPPH free radical scavenging rate of the enzymatic hydrolysate first increased and then decreased. There was no significant difference between initial pH 4 and initial pH 5 (P>0.05). The OH free radical scavenging rate of the enzymatic hydrolysate was better when the initial pH was between 3 and 5, and worse when the initial pH was 2 or 8. Considering the acid-base properties of royal jelly itself, the initial pH of 4 was selected for the next experiment.

[0054] 7. Orthogonal optimization of royal jelly enzymatic hydrolysis process On the basis of the above single factor experiment, the material-liquid ratio, enzymatic hydrolysis pH, enzymatic hydrolysis temperature and enzyme addition amount were used as experimental investigation factors, and the DPPH free radical scavenging rate and OH free radical scavenging rate were used as evaluation indicators to design L9 (4 3 ) Orthogonal experiment was used to optimize the extraction conditions (enzymatic hydrolysis time was 5 hours). The factors and levels design and experimental results are shown in Tables 1 and 2.

[0055]

[0056]

[0057] The results of the orthogonal optimization experiment of DPPH radical scavenging rate are shown in Table 1. From the R value analysis, it can be seen that the primary and secondary effects of the solid-liquid ratio, initial pH value and enzymatic hydrolysis temperature on the DPPH radical scavenging rate of the enzymatic hydrolyzate are: A>B>C>D, that is, the solid-liquid ratio has the greatest impact, followed by the initial pH value and enzymatic hydrolysis temperature, and the amount of enzyme added has the least effect on the DPPH radical scavenging rate of the enzymatic hydrolyzate. The final optimized combination is A1B2C3D3, that is, when the solid-liquid ratio is 1:4, the initial pH is 5, the enzymatic hydrolysis temperature is 50 ℃, and the enzyme addition amount is 9000 U / g, the enzymatic hydrolysis effect is the best.

[0058] The results of the orthogonal optimization experiment of OH radical scavenging rate are shown in Table 2. From the R value analysis, it can be seen that the primary and secondary effects of the solid-liquid ratio, initial pH value and enzymatic hydrolysis temperature on the OH radical scavenging rate of the enzymatic hydrolyzate are: A>D>C>B, that is, the solid-liquid ratio has the greatest impact, followed by the amount of enzyme added and the enzymatic hydrolysis temperature, and the initial pH has the least effect on the OH radical scavenging rate of the enzymatic hydrolyzate. The final optimized combination is A1B1C1D3, that is, when the solid-liquid ratio is 1:4, the initial pH is 4, the enzymatic hydrolysis temperature is 45 ℃, and the enzyme amount is 9000 U / g, the enzymatic hydrolysis effect is the best.

[0059] Compared with the product obtained at the initial pH 4, the royal jelly hydrolysate obtained at the initial pH 5 increased the scavenging rate of DPPH by 2%, and the scavenging rate of OH free radicals decreased by 1%. Factor B ranked second in the order of influence on the scavenging of DPPH by royal jelly hydrolysate, and fourth in the order of influence on the scavenging of OH free radicals. Therefore, the initial pH was finally determined to be 5. Compared with the product obtained at the temperature of 45℃, the royal jelly hydrolysate obtained at the temperature of 50℃ increased the scavenging rate of DPPH by 2%, and the scavenging rate of OH free radicals decreased by 3%. Factor C ranked third in the order of influence on the scavenging of DPPH and OH free radicals by royal jelly hydrolysate, so the temperature was finally determined to be 45℃.

[0060] After the above verification experiments, when the enzymatic hydrolysis conditions were: solid-liquid ratio 1:4, initial pH 5, enzymatic hydrolysis temperature 45°C, enzyme addition amount 9000 U / g, and enzymatic hydrolysis time 5 h, the scavenging rates of DPPH radicals and OH radicals were the highest, reaching 93% and 91% respectively.

[0061] Example 2 Optimization of gel filtration chromatography conditions and analysis of component activities 1. Molecular weight determination of enzymatic hydrolysate The molecular weight distribution was determined by high performance liquid chromatography. The chromatographic conditions were as follows: chromatographic column, TSK gel 2000SWXL 300 mm×7.8 mm; mobile phase, V (acetonitrile): V (water): V (trifluoroacetic acid) = 45:55:0.1; detection wavelength UV 220 nm; flow rate 0.5 mL / min; column temperature 30°C.

[0062] The enzymatic hydrolysate prepared in Example 1.7 (material-liquid ratio 1:4, initial pH 5, enzymatic hydrolysis temperature 45°C, enzyme addition 9000 U / g, enzymatic hydrolysis time 5 h) was used as the sample. The sample to be tested was diluted with the mobile phase and filtered through a 0.22 μm microporous filter membrane before injection. The molecular weight standards for the gel filtration column are: cytochrome C (12384 Da), porcine insulin (5733.49 Da), bacitracin (1422.69 Da), and glutathione (307.32 Da). A standard curve was drawn according to the peak time of the standard product, and the molecular weight of the royal jelly enzymatic hydrolysate polypeptide was calculated by fitting the drawn standard curve. The results are shown in Table 3. The molecular weight range is below 727 Da, so Sephadex G-15 was selected as the filler for the size exclusion chromatography column.

[0063]

[0064] 2. Analysis of the influence of different elution flow rates on purification effect The filler was swelled in excess deionized water at room temperature for 3 hours. During the swelling process, the upper layer of broken rubber was removed. The swollen filler, all buffers and other materials were balanced to the experimental operating temperature.

[0065] Wet the inside and bottom of the column with water and keep the liquid level for a short time to ensure that there are no bubbles at the bottom. Use a glass rod to guide the homogenate along the inner wall of the column and pour it into the column at one time. Be careful not to generate bubbles. Open the column outlet to allow the gel to settle freely in the column and connect the top of the column.

[0066] Before loading the sample, equilibrate the column for 5 column volumes until the baseline of the recorder becomes stable. The sample must be loaded after centrifugation filtration (0.45 μm).

[0067] The enzymatic hydrolysate prepared in Example 1.7 was used as a sample for loading, and the sample was filtered through a 0.45 μm filter membrane before loading. The loading concentration and volume were: 200 mg / mL, 2 mL; the peak components were collected, and the mobile phase was ultrapure water.

[0068] The appropriate elution flow rate will improve the separation effect of Sephadex G-15. When the sample load and sample concentration remain unchanged, the effects of four different elution flow rates on the separation effect were investigated: 1.5 mL / min, 3.5 mL / min, 5.5 mL / min, and 7.5 mL / min.

[0069] The results are as follows Figure 7 , Figure 8 , Fig. 9 , Fig.10As shown. When the elution flow rate is 1.5 mL / min, the flow rate is low, the sample fluidity is poor, the diffusion is aggravated, and the peak shape is not good. When the flow rate is 3.5 mL / min, the peak shape is improved. When the elution flow rate is 5.5 mL / min, the peak shape is better. When the elution flow rate is 7.5 mL / min, the flow rate is high, the small molecule peptides are eluted before entering the gel medium, and there is a tailing phenomenon between the peaks. Therefore, considering the peak shape comprehensively, it was finally determined to use a flow rate of 3.5 mL / min for the experiment.

[0070] 3. Analysis of the influence of different loading rates on purification effect Different sample loading volumes affect the separation effect of the Sephadex G-15 column. When the sample loading concentration and elution flow rate remain unchanged, the effects of 0.5 mL, 1 mL, and 1.5 mL sample loading volumes on the separation effect are investigated. The results are shown in Figure 2. Fig.11 , Fig.12 , Fig.13 When the sample volume was 0.5 mL, the content of each component after separation was small, resulting in poor peak shape; when the sample volume was 1.5 mL, tailing occurred due to the large amount of sample. Considering the peak shape and peak number comprehensively, a sample volume of 1 mL was finally selected for subsequent experiments.

[0071] 4. Optimal elution curve and determination results of each component like Fig.14 As shown, elution peaks F1 to F5 (the components obtained from each elution peak were named F1 to F5), were lyophilized and reconstituted into a solution with a polypeptide concentration of 24 mg / mL, and the DPPH scavenging rate and OH free radical scavenging rate of each component were determined. The results are shown in Fig.15A , Fig. 15B As shown in the figure (components 1 to 5 are F1 to F5 respectively). The scavenging rate of DPPH by F4 at a concentration of 24 mg / mL can reach 86%, and the scavenging rate of OH free radicals by F2 at a concentration of 24 mg / mL can reach 59%.

[0072] Example 3 LC-MS / MS mass spectrometry identification of F4 peptide composition 1. Peptide Extraction The sample (F4 obtained in Example 2) was re-dissolved by adding an appropriate amount of 0.1% TFA (trifluoroacetic acid) solution, and the OD 280 nm Determine peptide concentrations in preparation for LC-MS analysis.

[0073] 2. LC-MS / MS Analysis An appropriate amount of peptides was taken from each sample and chromatographically separated using a nanoliter flow rate Easy nLC 1200 chromatography system (ThermoScientific). Buffer: Solution A was a 0.1% formic acid aqueous solution, and solution B was a mixed solution of 0.1% formic acid, acetonitrile, and water (of which acetonitrile was 80%). The chromatographic column was equilibrated with 100% solution A. The sample was injected into a Trap Column (100 μm × 20 mm, 5 μm, C 18, Dr. Maisch GmbH) and then passed through a chromatographic analysis column (75 μm × 150 mm, 3 μm, C 18, Dr. Maisch GmbH) for gradient separation at a flow rate of 300 nl / min.

[0074] The liquid phase separation gradient is as follows: from 0 min to 2 min, the linear gradient of liquid B is from 2% to 5%; from 2 min to 44 min, the linear gradient of liquid B is from 5% to 28%; from 44 min to 51 min, the linear gradient of liquid B is from 28% to 40%; from 51 min to 53 min, the linear gradient of liquid B is from 40% to 100%; from 53 min to 60 min, liquid B is maintained at 100%.

[0075] After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive Plus mass spectrometer (Thermo Scientific). The analysis time was 60 min, detection mode: positive ion, parent ion scan range: 350-1800 m / z, primary mass spectrometry resolution: 60000 @ m / z 200, AGC target: 3e6, primary Maximum IT: 50 ms.

[0076] The peptide secondary mass spectrometry analysis was collected according to the following method: after each full scan (fμll scan), the secondary mass spectra (MS2 scan) of the 20 highest intensity parent ions were triggered to collect, the secondary mass spectrometry resolution: 15000 @m / z 200, AGCtarget: le5, secondary Maximum IT: 50 ms, MS2 Activation Type: HCD, Isolation window: 1.6 m / z, Normalized collisionenergy: 28.

[0077] 3. Database search The mass spectrometry database search software used in this project is Pfind. The following protein database is used: UniprotProtein Database. The analysis parameter settings of Pfind database search software are shown in Table 4.

[0078]

[0079] After mass spectrometry data retrieval, PSM FDR≤0.01 and Protein FDR≤0.01 were used as screening criteria for peptide, site and protein identification, respectively. 821 peptide sequences and 57 protein sequences were obtained. The sample mass spectrum Basepeak diagram is shown in the figure below: Fig.16 shown.

[0080] Example 4 Peptide screening, synthesis and verification 1. Peptide activity prediction According to the prediction scores of peptide biological activity by Peptide Raker, 36 peptides among 821 peptides have a prediction score greater than 0.8. At present, the quantitative structure-activity relationship of antioxidant peptides has not been fully elucidated, but a large number of research statistical results have found that the amino acid composition and its position in the peptide sequence play a vital role in the antioxidant activity of peptides: the amino acid residues (Tyr, Phe, Trp, Pro) at the N-terminus of the active peptide and the methionine at the C-terminus may be the key sites for free radical scavenging. The Pro at the N-terminus can provide protons to the DPPH free radical, thereby preventing the free radical chain reaction. The methionine sulfoxide, the oxidation product of Met at the C-terminus, has a significant antioxidant effect. The thiol group of the S atom on the Met in the sulfur-containing amino acid enables it to scavenge free radicals. In addition, Leu, Val, Ala, etc. can be used as hydrogen donors for free radical peroxidation on the side chain of aromatic residues to improve the antioxidant activity of peptides. Screening based on amino acid characteristics, a total of 15 peptides out of 36 peptides meet the above characteristics. The SVM scores of 15 potential physiologically active peptides were determined using the ToxinPred online tool, and all of them were negative, indicating that they were non-toxic.

[0081] After comprehensive consideration, the peptides that met the amino acid characteristics and were in the top 10 predicted scores by Peptide Ranker (FDRIW, YPDWSF, YPDWSW, FPYQPP, YPDWY, FNFDDVNFRIL, YPDWSFA, WHDKIF, WISPLY, and YPDWSWT) were screened for synthesis and verification of DPPH free scavenging activity.

[0082] 2. Peptide Synthesis The peptides FDRIW, YPDWSF, YPDWSW, FPYQPP, YPDWY, FNFDDVNFRIL, YPDWSFA, WHDKIF, WISPLY, and YPDWSWT were synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method.

[0083] 3. Activity Verification The peptides were prepared into a solution with a final concentration of 3.3 mg / mL, and the in vitro DPPH free radical scavenging rate of the synthetic peptides was determined according to the method of Example 1. The results are shown in Table 5. Among the 10 peptides, FDRIW, YPDWSW, and WHDKIF had the strongest activity, and the peptide free radical scavenging rate IC 50 As shown in Table 6, the mass spectrum is Fig.17 , 18 , as shown in 19.

[0084]

[0085]

[0086] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A small molecule peptide FDRIW, characterized by: Its amino acid sequence is shown in SEQ ID NO.

1.

2. Use of the small molecule peptide FDRIW according to claim 1 in the preparation of a health product with antioxidant activity, or in the preparation of a drug with the effect of inhibiting oxidative stress.

3. The use according to claim 2, characterized in that: The antioxidant activity is DPPH free radical scavenging activity.

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