A small molecule peptide with antioxidant activity, FDRIW, and its applications.

Abstract

This invention discloses a small molecule peptide, FDR1W, with antioxidant activity and its applications, belonging to the field of enzymatic hydrolysis processing technology. The amino acid sequence of the FDR1W is shown in SEQ ID NO.1. The FDR1W is used in the preparation of health products with antioxidant activity and in the preparation of drugs with inhibitory oxidative stress effects. This invention screened and obtained a small molecule peptide with high scavenging activity against DPPH free radicals, IC50. 50 =3.63 mg / ml, which has the potential to be used as an antioxidant functional product.

Description

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

[0002] This invention relates to a small molecule peptide, FDRIW, with antioxidant activity and its applications, belonging to the technical field of enzymatic hydrolysis products. Background Technology

[0003] Royal jelly is a secretion from the hypopharyngeal and mandibular glands of young worker bees. It is composed of water, protein, carbohydrates, lipids, minerals, and vitamins, and has high nutritional value. It is widely used in food, biology, and medicine, possessing various physiological functions such as anti-inflammation, antibacterial properties, anti-tumor activity, and immune enhancement. However, its processing and utilization are very limited, resulting in low economic added value.

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

[0005] Oxidative stress is a contributing factor to atherosclerosis, cardiovascular disease, cancer, diabetes, and neurodegenerative diseases. The body's naturally occurring antioxidant system is often insufficient to meet the body's needs under external stimuli, requiring additional antioxidants through diet or medication to reduce oxidative stress and achieve effects such as delaying aging, improving sub-health conditions, and reducing the incidence of disease. Synthetic antioxidants such as BHA, BHT, and gallic acid have good antioxidant effects; however, these substances have certain side effects. Natural, safe antioxidants are currently a hot research topic. 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 its application.

[0007] This invention is achieved through the following technical solution:

[0008] A small molecule peptide, FDR1W, with antioxidant activity, has the amino acid sequence shown in SEQ ID NO.1.

[0009] The aforementioned small molecule peptides have good DPPH free radical scavenging activity and can be used to prepare health products with antioxidant activity and drugs with the function of inhibiting oxidative stress.

[0010] This invention processes royal jelly using a specific method (enzymatic hydrolysis + gel filtration chromatography) to obtain royal jelly peptides with antioxidant activity. LC-MS / MS identification and screening revealed three small peptides with high scavenging activity against DPPH free radicals: FDR1W (IC50, 1000 ppm). 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.

[0011] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0012] Figure 1A Schematic diagram of the effect of protease on DPPH free radical scavenging rate.

[0013] Figure 1B Schematic diagram of the effect of protease on OH free radical scavenging rate.

[0014] Figure 2A Schematic diagram of the effect of enzymatic hydrolysis time on DPPH free radical scavenging rate.

[0015] Figure 2B Schematic diagram of the effect of enzymatic hydrolysis time on OH free radical scavenging rate.

[0016] Figure 3A Schematic diagram of the effect of temperature on DPPH free radical scavenging rate.

[0017] Figure 3B Schematic diagram of the effect of temperature on OH radical scavenging rate.

[0018] Figure 4A Schematic diagram of the effect of enzyme dosage on DPPH free radical scavenging rate.

[0019] Figure 4B Schematic diagram of the effect of enzyme dosage on OH free radical scavenging rate.

[0020] Figure 5A Schematic diagram of the effect of feed-to-liquid ratio on DPPH free radical scavenging rate.

[0021] Figure 5B Schematic diagram of the effect of feed-to-liquid ratio on OH radical scavenging rate.

[0022] Figure 6A Schematic diagram of the effect of initial pH value on DPPH free radical scavenging rate.

[0023] Figure 6B Schematic diagram of the effect of initial pH value on OH radical scavenging rate.

[0024] Figure 7 Schematic diagram of the effect of a flow rate of 1.5 mL / min on the separation performance of Sephadex G-15.

[0025] Figure 8 Schematic diagram showing the effect of a flow rate of 3.5 mL / min on the separation performance of Sephadex G-15.

[0026] Figure 9 Schematic diagram showing the effect of a flow rate of 5.5 mL / min on the separation performance of Sephadex G-15.

[0027] Figure 10 Schematic diagram showing the effect of a flow rate of 7.5 mL / min on the separation performance of Sephadex G-15.

[0028] Figure 11 Schematic diagram showing the effect of 0.5 mL sample loading on the separation effect of Sephadex G-15.

[0029] Figure 12 Schematic diagram showing the effect of 1 mL sample loading on the separation effect of Sephadex G-15.

[0030] Figure 13 Schematic diagram showing the effect of 1.5 mL sample loading on the separation effect of Sephadex G-15.

[0031] Figure 14 Schematic diagram of the optimal elution curve for Sephadex G-15.

[0032] Figure 15A Schematic diagram of the DPPH free radical scavenging rate of each component.

[0033] Figure 15B Schematic diagram of the results of the determination of the OH free radical scavenging rate of each component.

[0034] Figure 16 : Sample mass spectrometry Basepeak plot.

[0035] Figure 17 : Mass spectrum of FDRIVE.

[0036] Figure 18 Mass spectrum of YPDWSW.

[0037] Figure 19 : Mass spectrum of WHDKIF. Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0039] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0040] The royal jelly used in the following examples was purchased from Shandong Fengcai Health Industry Co., Ltd., and placed in a sealed, opaque bag at -20°C for later use.

[0041] Example 1: Single-factor optimization of royal jelly enzymatic hydrolysis process

[0042] 1. Enzyme screening

[0043] Papain, bromelain, neutral protease, alkaline protease, and acidic protease were selected for enzymatic hydrolysis of the raw materials. 2 g of pre-frozen royal jelly was accurately weighed and prepared as a substrate solution at a material-to-liquid ratio of 1:8. Five proteases were added at an enzyme dosage of 4000 U / g (enzyme activity / royal jelly). After reacting at 50℃ and 200 rpm for 4 h, samples were taken, enzymes were inactivated, the mixture was cooled, and centrifuged (8000 rpm, 10 min). The supernatant was collected, and the DPPH free radical scavenging rate and OH free radical scavenging rate were determined.

[0044] Determination of DPPH free radical scavenging rate: The spectrophotometer / microplate reader was preheated for 30 min, and the wavelength was adjusted to 517 nm. 500 μl of the enzyme digest was mixed with 1000 μl of 0.2 mM DPPH solution (dissolved in anhydrous ethanol), shaken well, and reacted in the dark at room temperature for 20 min, then filtered. The absorbance value Ai at 517 nm was measured. The absorbance value Aj was measured using 500 μl of anhydrous ethanol instead of the DPPH solution, and the absorbance value A0 was measured using 500 μl of water instead of the enzyme digest. The DPPH free radical scavenging rate was calculated as: DPPH scavenging rate = (1 - Ai - Aj / A0) × 100%. BHA (butylated hydroxyanisole) was used as a control.

[0045] 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 in the dark at 37℃ for 30 min. Measure the absorbance Ai at 510 nm. Simultaneously, replace 0.5 mL of H2O2 solution with 0.5 mL of distilled water and measure its absorbance Aj; replace 0.1 mL of the sample solution with 0.1 mL of distilled water and measure its absorbance A0. The formula for calculating the OH radical scavenging rate is: OH radical scavenging rate = (1 - Ai - Aj / A0) × 100%. Vitamin C was used as a control.

[0046] This experiment selected five proteases to enzymatically hydrolyze royal jelly, and the results are as follows: Figure 1A , Figure 1B As shown in the figure, papain, bromelain, neutral protease, alkaline protease, and acidic protease were used to enzymatically hydrolyze royal jelly. In the DPPH free radical scavenging experiment, acidic protease showed the best effect, followed by alkaline protease and neutral protease. In the OH free radical scavenging experiment, acidic protease, alkaline protease, and neutral protease all showed good free radical scavenging effects. Considering factors such as protease efficacy and addition cost, acidic protease was selected as the enzyme preparation for further optimization.

[0047] 2. Optimization of enzymatic hydrolysis time

[0048] Accurately weigh 2 g of pre-frozen royal jelly and prepare a substrate solution at a material-to-liquid ratio of 1:8. Add acidic protease at an enzyme dosage of 4000 U / g and adjust the initial pH of the substrate solution to 4. After reacting 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, using the same method as above.

[0049] The results are as follows Figure 2A , Figure 2B As shown in the figure, DPPH free radical scavenging activity increased with increasing enzymatic hydrolysis time in the short term. A significant difference was found between 3 or 4 hours of hydrolysis and 5 hours of hydrolysis (P<0.05), while no significant difference was found between 5–8 hours of hydrolysis (P>0.05). OH free radical scavenging activity was highest at 5 hours of hydrolysis, showing a significant difference from other groups (P<0.05). Considering both the hydrolysis effect and efficiency, 5 hours was ultimately selected as the hydrolysis treatment time.

[0050] 3. Optimization of enzymatic hydrolysis temperature

[0051] Accurately weigh 2 g of pre-frozen royal jelly and prepare a substrate solution at a material-to-liquid ratio of 1:8 with an initial pH of 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. Take samples, inactivate the enzyme, cool, and centrifuge (8000 rpm, 10 min) to collect the supernatant. Measure the DPPH free radical scavenging rate and OH free radical scavenging rate using the same method as above.

[0052] The results are as follows Figure 3A , Figure 3B As shown, the DPPH radical scavenging activity initially increased and then decreased with increasing enzymatic hydrolysis temperature. The highest DPPH radical scavenging activity was observed at 50℃, showing no significant difference from 60℃ (P>0.05), but significantly higher than other groups (P<0.05). OH radical scavenging activity did not change significantly with temperature. Considering the overall enzymatic hydrolysis effect, 50℃ was selected as the enzymatic hydrolysis treatment temperature.

[0053] 4. Optimization of enzyme dosage

[0054] Accurately weigh 2 g of pre-frozen royal jelly and prepare a substrate solution at a material-to-liquid ratio of 1:8. Add acidic protease at enzyme dosages of 2000, 4000, 6000, 8000, and 10000 U / g. React at 50℃ and 200 rpm for 5 h, then take a sample, inactivate the enzyme, cool, centrifuge (8000 rpm, 10 min), and collect the supernatant. Measure the DPPH free radical scavenging rate and OH free radical scavenging rate using the same method as above.

[0055] The results are as follows Figure 4A , Figure 4B As shown, with increasing enzyme dosage, the DPPH radical scavenging rate of the enzymatic hydrolysate generally showed a gradually increasing trend. There was no significant difference between enzyme dosages of 6000 U / g and 8000 U / g (P>0.05); however, there was a significant difference between enzyme dosages of 8000 U / g and 10000 U / g (P<0.05). The OH radical scavenging rate of the enzymatic hydrolysate was not significantly affected by the enzyme dosage. Considering overall cost, an enzyme dosage of 8000 U / g was selected for further experiments.

[0056] 5. Optimization of feed-liquid ratio

[0057] Accurately weigh 2 g of pre-frozen royal jelly and prepare substrate solutions at material-to-liquid ratios of 1:3, 1:5, 1:7, 1:9, and 1:11, respectively. Add acidic protease at an enzyme dosage of 8000 U / g and react at 50℃ and 200 rpm for 5 h. After reaction, take samples, inactivate the enzyme, cool, and centrifuge (8000 rpm, 10 min) to collect the supernatant. Measure the DPPH free radical scavenging rate and OH free radical scavenging rate using the same method as above.

[0058] The results are as follows Figure 5A , Figure 5B As shown, the DPPH and OH radical scavenging rates of the enzymatic hydrolysate gradually decreased with increasing material-to-liquid ratio. There was no significant difference in DPPH radical scavenging rate between a material-to-liquid ratio of 1:5 and 1:3 (P>0.05). However, there was a significant difference in OH radical scavenging rate between the two ratios (P<0.05). Considering all factors, a material-to-liquid ratio of 1:3 was selected for the next stage of the experiment.

[0059] 6. Initial pH optimization

[0060] Accurately weigh 2 g of pre-frozen royal jelly and prepare a substrate solution at a material-to-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 acidic protease at an enzyme dosage of 8000 U / g. After reacting at 50℃ and 200 rpm for 5 h, take a sample, inactivate the enzyme, cool, and 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.

[0061] The results are as follows Figure 6A , Figure 6B As shown, with increasing initial pH, the DPPH free radical scavenging rate of the enzymatic hydrolysate initially 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 between initial pH 3 and 5, and worse at initial pH 2 or 8. Considering the acid-base properties of royal jelly itself, an initial pH of 4 was selected for the next stage of the experiment.

[0062] 7. Orthogonal optimization of royal jelly enzymatic hydrolysis process

[0063] Based on the above single-factor experiments, with the material-to-liquid ratio, enzymatic hydrolysis pH, enzymatic hydrolysis temperature, and enzyme dosage as the experimental factors, and DPPH free radical scavenging rate and OH free radical scavenging rate as the evaluation indicators, an L9(4)24 ... 3 The extraction conditions (enzymatic hydrolysis time of 5 hours) were optimized by orthogonal experiments. The factor and level design and experimental results are shown in Table 1 and Table 2.

[0064]

[0065]

[0066] The results of the orthogonal optimization experiment on DPPH free radical scavenging rate are shown in Table 1. Analysis of the R-values ​​revealed that the order of influence of the material-to-liquid ratio, initial pH, and enzymatic hydrolysis temperature on the DPPH free radical scavenging rate of the enzymatic hydrolysate was: A>B>C>D. That is, the material-to-liquid ratio had the greatest impact, followed by the initial pH and enzymatic hydrolysis temperature, while the enzyme dosage had the least impact on the DPPH free radical scavenging rate. The final optimized combination was A1B2C3D3, meaning that the best enzymatic hydrolysis effect was achieved when the material-to-liquid ratio was 1:4, the initial pH was 5, the enzymatic hydrolysis temperature was 50 ℃, and the enzyme dosage was 9000 U / g.

[0067] The results of the orthogonal optimization experiment on OH radical scavenging rate are shown in Table 2. Analysis of the R-values ​​revealed that the order of influence of the material-to-liquid ratio, initial pH, and enzymatic hydrolysis temperature on the OH radical scavenging rate of the hydrolysate was: A>D>C>B, meaning the material-to-liquid ratio had the greatest impact, followed by the enzyme dosage and hydrolysis temperature. The initial pH had the least impact on the OH radical scavenging rate. The final optimized combination was A1B1C1D3, indicating that the optimal enzymatic hydrolysis effect was achieved when the material-to-liquid ratio was 1:4, the initial pH was 4, the hydrolysis temperature was 45 ℃, and the enzyme dosage was 9000 U / g.

[0068] Compared to the product obtained at an initial enzyme pH of 4, the royal jelly enzymatic hydrolysate obtained at an initial pH of 5 showed a 2% increase in DPPH scavenging rate and a 1% decrease in OH radical scavenging rate. Factor B ranked second in its influence on DPPH scavenging and fourth in its influence on OH radical scavenging. Therefore, the initial pH was ultimately determined to be 5. Compared to the product obtained at 45℃, the royal jelly enzymatic hydrolysate obtained at 50℃ showed a 2% increase in DPPH scavenging rate and a 3% decrease in OH radical scavenging rate. Factor C ranked third in its influence on both DPPH and OH radical scavenging; therefore, the final temperature was determined to be 45℃.

[0069] Based on the above verification experiments, the highest DPPH radical and OH radical scavenging rates were achieved under the following conditions: material-to-liquid ratio 1:4, initial pH 5, enzymatic hydrolysis temperature 45℃, enzyme dosage 9000 U / g, and enzymatic hydrolysis time 5 h, reaching 93% and 91%, respectively.

[0070] Example 2 Optimization of gel filtration chromatography conditions and analysis of the activity of its components

[0071] 1. Determination of molecular weight of enzymatic hydrolysate

[0072] The molecular weight distribution was determined by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: 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℃.

[0073] The enzymatic hydrolysate prepared in Example 1.7 (solid-to-liquid ratio 1:4, initial pH 5, hydrolysis temperature 45℃, enzyme dosage 9000 U / g, hydrolysis time 5 h) was used as the sample. The sample was diluted with the mobile phase, filtered through a 0.22 μm microporous membrane, and then injected. The molecular weight standards for the gel filtration chromatography column were: cytochrome C (12384 Da), porcine insulin (5733.49 Da), bacitracin (1422.69 Da), and glutathione (307.32 Da). A standard curve was plotted based on the peak times of the standards. The molecular weight of the peptides in the royal jelly enzymatic hydrolysate was calculated by fitting the standard curve. The results are shown in Table 3. The molecular weight range was below 727 Da; therefore, Sephadex G-15 dextran gel was selected as the packing material for the size exclusion chromatography column.

[0074]

[0075] 2. Analysis of the effect of different elution flow rates on purification efficiency

[0076] The packing material was swelled in excess deionized water at room temperature for 3 hours. During the swelling process, the upper layer of broken gel was removed. The swollen packing material and all buffer solutions and other materials were then equilibrated to the experimental operating temperature.

[0077] Wet the inside and bottom of the column with water, maintaining a small level to ensure no air bubbles at the bottom. Use a glass rod to guide the homogenate along the inner wall of the column in one pour, being careful not to create air bubbles. Open the column outlet to allow the gel to settle freely within the column, and secure the top of the column.

[0078] Before loading the sample, equilibrate the column to 5 column volumes until the recorder baseline becomes stable. The sample must be loaded after filtration (0.45 μm).

[0079] The enzymatic hydrolysate prepared in Example 1.7 was used as the sample for loading, and filtered through a 0.45 μm filter membrane before loading. The loading concentration and volume were 200 mg / mL, 2 mL; the eluting components were collected, and the mobile phase was ultrapure water.

[0080] An appropriate elution flow rate can improve the separation performance of Sephadex G-15. The effects of four different elution flow rates (1.5 mL / min, 3.5 mL / min, 5.5 mL / min, and 7.5 mL / min) on the separation performance were investigated, with constant sample loading volume and concentration.

[0081] The results are as follows Figure 7 , Figure 8 , Figure 9 , Figure 10 As shown, at an elution flow rate of 1.5 mL / min, the flow rate was too low, resulting in poor sample flowability, increased diffusion, and undesirable peak shape. The peak shape improved at a flow rate of 3.5 mL / min. The peak shape was also good at an elution flow rate of 5.5 mL / min. At an elution flow rate of 7.5 mL / min, the flow rate was too high, causing small molecule peptides to be eluted before entering the gel medium, and tailing occurred between peaks. Therefore, considering both peak shape and flow rate, a flow rate of 3.5 mL / min was ultimately selected for the experiment.

[0082] 3. Analysis of the effect of different sample loading rates on purification efficiency

[0083] Different loading volumes affect the separation performance of the Sephadex G-15 column. With the loading concentration and elution flow rate kept constant, the effects of loading volumes of 0.5 mL, 1 mL, and 1.5 mL on the separation performance were investigated. The results are as follows: Figure 11 , Figure 12 , Figure 13 As shown, when the sample loading volume was 0.5 mL, the concentration of each component after separation was low, resulting in poor peak shape; when the sample loading volume was 1.5 mL, tailing occurred due to the large sample size. Considering both peak shape and peak number, a sample loading volume of 1 mL was ultimately selected for subsequent experiments.

[0084] 4. Optimal elution curve and determination results of each component

[0085] like Figure 14 As shown, elution peaks F1–F5 were collected sequentially (the fractions obtained from each elution peak were named F1–F5 respectively), lyophilized, and reconstituted into a solution with a peptide concentration of 24 mg / mL. The DPPH scavenging rate and OH radical scavenging rate of each fraction were measured, and the results are shown below. Figure 15A , Figure 15B As shown in the figure (components 1-5 are F1-F5 respectively). F4 can achieve a DPPH scavenging rate of 86% at a concentration of 24 mg / mL, and F2 can achieve an OH radical scavenging rate of 59% at a concentration of 24 mg / mL.

[0086] Example 3: LC-MS / MS mass spectrometry identification of F4 peptide composition

[0087] 1. Peptide extraction

[0088] The sample (F4 obtained in Example 2) was reconstituted with an appropriate amount of 0.1% TFA (trifluoroacetic acid) solution, and the OD... 280 nm Peptide concentrations were determined for LC-MS analysis.

[0089] 2. LC-MS / MS analysis

[0090] A suitable amount of peptide was taken from each sample and chromatographically separated using a Nano-flow-rate Easy nLC 1200 chromatography system (ThermoScientific). Buffer solutions: Solution A was a 0.1% formic acid aqueous solution, and Solution B was a mixture of 0.1% formic acid, acetonitrile, and water (acetonitrile comprising 80%). The column was equilibrated with 100% Solution A. After injection into a TrapColumn (100 μm × 20 mm, 5 μm, C 18, Dr. Maisch GmbH), the sample underwent gradient separation using an analytical column (75 μm × 150 mm, 3 μm, C 18, Dr. Maisch GmbH) at a flow rate of 300 nL / min.

[0091] The liquid phase separation gradients are as follows: from 0 to 2 minutes, the linear gradient of liquid B increases from 2% to 5%; from 2 to 44 minutes, the linear gradient of liquid B increases from 5% to 28%; from 44 to 51 minutes, the linear gradient of liquid B increases from 28% to 40%; from 51 to 53 minutes, the linear gradient of liquid B increases from 40% to 100%; and from 53 to 60 minutes, liquid B remains at 100%.

[0092] 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, precursor ion scan range: 350–1800 m / z, primary mass spectrometry resolution: 60000 m / z 200, AGC target: 3e6, primary maximum IT: 50 ms.

[0093] Peptide secondary mass spectrometry analysis was performed using the following method: after each full scan (fμll scan), the secondary mass spectra of the 20 highest intensity precursor ions were acquired (MS2 scan). Secondary mass spectrometry resolution: 15000 m / z 200, AGC target: le5, secondary maximum IT: 50 ms, MS2 activation type: HCD, isolation window: 1.6 m / z, normalized collision energy: 28.

[0094] 3. Database retrieval

[0095] The mass spectrometry database search software used in this project is Pfind. The following protein database was used: Uniprot Protein Database. The Pfind search software analysis parameter settings are shown in Table 4.

[0096]

[0097] 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 spectrometry basepeak plots are shown below. Figure 16 As shown.

[0098] Example 4: Peptide screening, synthesis, and validation

[0099] 1. Peptide activity prediction

[0100] Based on the Peptide Raker prediction scores for peptide bioactivity, 36 peptides out of 821 peptides had prediction scores greater than 0.8. While the quantitative structure-activity relationship (QS) of antioxidant peptides is not yet fully elucidated, numerous studies have revealed that amino acid composition and their positions within the peptide sequence play a crucial role in the peptide's antioxidant activity: N-terminal amino acid residues (Tyr, Phe, Trp, Pro) and C-terminal methionine residues may be key sites for free radical scavenging. The N-terminal Pro residue can donate protons to DPPH free radicals, thereby preventing free radical chain reactions. The C-terminal Met oxidation product, methionine sulfoxide, exhibits significant antioxidant effects; the thiol group on the S atom of the Met atom in sulfur-containing amino acids endows it with free radical scavenging capabilities. Furthermore, Leu, Val, and Ala can act as hydrogen donors for free radical peroxidation on aromatic residue side chains, enhancing the peptide's antioxidant activity. Screening based on amino acid characteristics, 15 out of the 36 peptides met the above characteristics. The SVM scores of 15 potentially physiologically active peptides were determined using the ToxinPred online tool, and all scores were negative, indicating that none of them were toxic.

[0101] Taking all factors into consideration, peptides that meet the amino acid characteristics and are among the top 10 in Peptide Ranker prediction scores (FDRIW, YPDWSF, YPDWSW, FPYQPP, YPDWY, FNFDDVNFRIL, YPDWSFA, WHDKIF, WISPLY, and YPDWSWT) were screened for synthesis and their DPPH free radical scavenging activity was verified.

[0102] 2. Peptide synthesis

[0103] The peptides FDR1W, YPDWSF, YPDWSW, FPYQPP, YPDWY, FNFDDVNFRIL, YPDWSFA, WHDKIF, WISPLY, and YPDWSWT were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. using the Fmoc solid-phase synthesis method.

[0104] 3. Activity verification

[0105] The peptides were prepared into solutions with a final concentration of 3.3 mg / mL, and the in vitro DPPH free radical scavenging rate of the synthesized peptides was determined according to the method in Example 1. The results are shown in Table 5. Among the 10 peptides, FDR1W, YPDWSW, and WHDKIF showed the strongest activity, with an IC50 of [missing value]. 50 As shown in Table 6, the mass spectra are as follows: Figure 17 , 18 As shown in Figure 19.

[0106]

[0107]

[0108] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

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

1.

2. The use of the small molecule peptide FDR1W according to claim 1 in the preparation of health products with antioxidant activity, or in the preparation of drugs with the function of inhibiting oxidative stress.

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

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