Soybean active peptide with antioxidant and anti-aging activity as well as preparation method and application of soybean active peptide

Abstract

The invention relates to a soybean active peptide with antioxidant and anti-aging activity and a preparation method and application thereof, and belongs to the technical field of active peptide protein. The active peptide is selected from SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; the amino acid sequences of the SEQ ID NO.1, the SEQ ID NO.2 and the SEQ ID NO.3 are VLLPGP, PAGLYP and FLVLPA respectively, and the amino acid sequences of the SEQ ID NO.1, the SEQ ID NO.2 and the SEQ ID NO.3 are shown in the description. Experiments prove that the soybean active peptide has antioxidant and anti-aging activity, is safe and free of toxic and side effects, can be used as a functional component in antioxidant and anti-aging cosmetics, medicines and the like, and has good potential and application prospects.

Description

Technical Field

[0001] The present invention relates to soybean active peptides with anti-oxidation and anti-aging activities, a preparation method and application thereof, and belongs to the technical field of active peptide proteins. Background Art

[0002] Peptides are fragments consisting of 2-20 amino acid residues produced by protein degradation. Based on their biological activity, peptides are categorized as antioxidant peptides, anti-aging peptides, anti-inflammatory peptides, antimicrobial peptides, glucose-lowering peptides, blood pressure-lowering peptides, lipid-lowering peptides, anticancer peptides, and immune peptides. Antioxidant peptides, defined as polypeptides with antioxidant activity, have broad potential applications in functional foods, pharmaceuticals, and cosmetics.

[0003] The generation and accumulation of free radicals in the human body contribute to aging and various diseases (such as arthritis, arteriosclerosis, coronary heart disease, neurological disorders, and cancer). Antioxidants exert their antioxidant effects by scavenging free radicals, inhibiting lipid peroxidation, and chelating metal ions, preventing and treating diseases and delaying aging. Commonly used chemically synthesized antioxidants include butylated hydroxyanisole, tert-butylhydroquinone, butylated hydroxytoluene, and propyl gallate. However, these chemicals pose certain safety risks. Antioxidant peptides, derived from food proteins, are garnering attention both domestically and internationally for their safety and effectiveness.

[0004] Soybeans contain approximately 40% protein, 20% oil, 35% carbohydrates, and 5% ash, making them highly nutritious. Soy protein is a high-quality protein with a high content of essential amino acids. The main components of soy protein are β-conglycinin and glycinin, which together account for 80%-90% of the total soy protein content. Soy protein degradation produces soy biopeptides. The main methods for producing soy biopeptides include enzymatic hydrolysis, microbial fermentation, and chemical processing. Microbial fermentation, which utilizes specific microorganisms and the proteases they produce to hydrolyze soy protein, offers advantages such as low production cost, mild reaction conditions, ease of operation, and environmental friendliness. Microbial fermentation can also improve the bioavailability, nutritional quality, and digestibility of functional components in soybeans, extend shelf life, improve sensory quality, increase beneficial active ingredients, and reduce anti-nutritional factors. Commonly used microorganisms for soy fermentation include bacteria such as Lactococcus, Lactobacillus, Streptococcus thermophilus, and Bacillus subtilis, as well as fungi such as Saccharomyces and Aspergillus niger. Soy peptides obtained by fermenting soybeans with different microorganisms have varying sequences and activities.

[0005] So far, active soybean peptides such as soy glucose-lowering peptides, soy immune peptides, soy anti-inflammatory peptides, soy blood pressure-lowering peptides, soy lipid-lowering peptides, soy anti-cancer peptides, and soy antioxidant peptides have been screened from soy protein degradation products. Reported soy antioxidant peptide sequences include NPESQQGSPRV, PGTAVFK, IKAFKEATKVDKVVVLWTA, VNPESQQGSPR, and LH. Summary of the Invention

[0006] In response to the problems existing in the above-mentioned prior art, the present invention provides soybean active peptides with antioxidant and anti-aging activities, as well as preparation methods and applications thereof. The soybean active peptides obtained by the present invention have the advantages of being safe and having no toxic side effects.

[0007] The technical solutions of the present invention are as follows: A soybean active peptide with antioxidant and anti-aging activities, wherein the active peptide is selected from SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3; the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are Val-Leu-Leu-Pro-Gly-Pro (VLLPGP), Pro-Ala-Gly-Leu-Tyr-Pro (PAGLYP) and Phe-Leu-Val-Leu-Pro-Ala (FLVLPA), respectively.

[0008] The preparation method of the above-mentioned soybean active peptide comprises the following steps: (1) Using soybean protein as raw material, distilled water was added at a material-liquid ratio of 1:20 (w / w), mixed, sterilized by high-pressure steam at 121°C for 15 min, and cooled to room temperature to obtain a sterilized soybean protein solution; (2) Probiotic liquid fermentation of soybean protein to prepare soybean active peptides: inoculate Bacillus natto into the sterilized soybean protein solution obtained in step (1), shake culture at 37°C for 24 h, and shake at a speed of 100-200 rpm; then inoculate Bacillus coagulans, shake culture at 47°C for 42 h, and shake at a speed of 100-200 rpm; then inoculate Lactobacillus rhamnosus, shake culture at 37°C for 36 h, and shake at a speed of 100-200 rpm; obtain fermentation liquid; Preferably, in step (2), the inoculation amount of Bacillus natto, Bacillus coagulans and Lactobacillus rhamnosus is 2% of the mass of the sterilized soy protein solution.

[0009] (3) The fermentation broth was ultrafiltered using an ultrafiltration centrifuge tube with a molecular weight cutoff of 1 kDa. The filtrate was centrifuged at 10,000-12,000 rpm for 15 min at 4°C, and the filtrate was collected as the soybean peptide solution, which was named SPAP2. (4) Filter SPAP2 with a 0.22 μm aqueous syringe filter to remove particles, and perform mass spectrometry identification using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-TOF-MS / MS) to obtain the soybean peptide sequence; Preferably, the mobile phase A of UPLC is an aqueous solution containing 0.1% formic acid, the mobile phase B is an acetonitrile solution containing 0.1% formic acid, the elution time is 60 min, the gradient elution conditions are shown in Table 1, the injection volume is 5 μL, and the flow rate is set to 300 μL / min.

[0010] Table 1 Ultra-high performance liquid chromatography elution conditions

[0011] Preferably, the mass spectrometry analysis adopts positive ion mode, secondary tandem mass spectrometry analysis, a resolution of 120,000, and a 60-2000 m / z full MS scan. The tandem mass spectrometry analysis conditions are shown in Table 2.

[0012] Table 2 Tandem mass spectrometry analysis conditions

[0013] (5) Bioinformatics prediction of each soybean peptide sequence: 1) Prediction of potential biological activity of peptides: Use PeptideRanker to predict and analyze the biological activity of peptides, and screen peptide sequences with PeptideRanker prediction values ​​greater than 0.5 for subsequent analysis; 2) Peptide toxicity prediction analysis: ToxinPrep, based on the SVM algorithm, was used to perform potential toxicity prediction analysis on peptide sequences with a PeptideRanker prediction value greater than 0.5 in step 1); 3) Prediction of antioxidant activity of soybean peptides: For soybean peptide sequences with PeptideRanker > 0.5 and no toxicity in step 2), AnOxPePred was used to predict their free radical scavenging capacity (FRS) and metal ion chelating capacity (CHEL). Peptide sequences with FRS values ​​> 0.5 or CHEL values ​​> 0.25 were screened; the soybean active peptides VLLPGP, PAGLYP, and FLVLPA were obtained.

[0014] The present invention also includes the use of the soybean active peptide in the preparation of antioxidant and anti-aging medicines or cosmetics.

[0015] Compared with the prior art, the present invention has the following advantages: The present invention screened and obtained three new peptides, VLLPGP, PAGLYP and FLVLPA, from soybean for the first time. The bioinformatics prediction results showed that VLLPGP, PAGLYP and FLVLPA had no toxic side effects and high biological activity. The results of in vitro antioxidant experiments showed that the ABTS free radical scavenging rate IC 50The values are 0.65 mM, 1.74 mM, and 0.79 mM, respectively. The in vivo antioxidant and anti-aging activities of the soybean peptides were evaluated on the model organism Caenorhabditis elegans. The oxidative stress test results showed that 1 mg / mL VLLPGP, PAGLYP, and FLVLPA solutions could increase the average lifespan of C. elegans under oxidative stress by 38.2%, 15.12%, and 27.58%, respectively. The anti-aging experiment results showed that 1 mg / mL peptide solutions VLLPGP, PAGLYP, and FLVLPA could extend the average lifespan of C. elegans by 44.03%, 23.46%, and 40.22%, respectively, which was significantly different from the control group. The in vivo antioxidant experiment confirmed that VLLPGP, PAGLYP, and FLVLPA could increase the activity of SOD and reduce the content of ROS, thereby exerting antioxidant and anti-aging effects. In summary, the in vitro and in vivo experimental results both confirmed that the new peptides VLLPGP, PAGLYP, and FLVLPA have antioxidant and anti-aging activities. Therefore, the new antioxidant and anti-aging soybean peptides VLLPGP, PAGLYP, and FLVLPA can be used as functional ingredients in antioxidant and anti-aging cosmetics, drugs, and the like, and have good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 ABTS free radical scavenging rate determination results of SPAP2 obtained in Example 1; Figure 2 Survival curves of nematodes treated with different concentrations of SPAP2. DETAILED DESCRIPTION

[0017] The advantages and characteristics of the present application will become more apparent with the description. However, the examples are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements all fall within the protection scope of the present application.

[0018] The Bacillus natto, Bacillus coagulans, and Lactobacillus rhamnosus used in the embodiments of the present application are all purchased from a strain preservation center. The Bacillus natto, classified as Bacillius subtilis, has a preservation number of CGMCC 4731 and is purchased from the China General Microbiological Culture Collection Center; the Bacillus coagulans, classified as Bacillus coagulans, has a preservation number of CCTCC AB 2018147 and is purchased from the China Center for Type Culture Collection; and the Lactobacillus rhamnosus, classified as Lactobacillus rhamnosus, has a preservation number of CICC21006 and is purchased from the China Industrial Microbial Culture Collection Center.

[0019] Example 1 A soybean active peptide with antioxidant and anti-aging activities, wherein the active peptide is selected from SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0020] The preparation method of the above-mentioned soybean active peptide comprises the following steps: (1) Using soy protein as raw material, distilled water was added at a material-liquid ratio of 1:20 (w / w), mixed, and sterilized by high-pressure steam at 121°C for 15 min. The temperature of the soy protein solution was allowed to cool to room temperature. (2) The sterilized soy protein solution was inoculated with probiotics for liquid fermentation to prepare soy active peptides: first, Bacillus natto was inoculated (inoculation amount was 2%, w / w), and cultured at 37°C with shaking for 24 h, and the shaker speed was 180 rpm; then, Bacillus coagulans was inoculated (inoculation amount was 2%, w / w), and cultured at 47°C with shaking for 42 h, and the shaker speed was 180 rpm; then, Lactobacillus rhamnosus was inoculated (inoculation amount was 2%, w / w), and cultured at 37°C with shaking for 36 h, and the shaker speed was 180 rpm; (3) The fermentation broth was ultrafiltered using an ultrafiltration centrifuge tube with a molecular weight cutoff of 1 kDa. The ultrafiltration fraction with a molecular weight less than 1 kDa was collected at 10,000 rpm for 15 min at 4°C, and the soy peptide solution was named SPAP2. (4) Mass spectrometry identification of SPAP2: 1) Filter SPAP2 through a 0.22 μm aqueous syringe filter to remove particulates. Mass spectrometric identification was then performed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-TOF-MS / MS). The UPLC mobile phase A consisted of aqueous solution (containing 0.1% formic acid) and mobile phase B consisted of acetonitrile (containing 0.1% formic acid). The elution time was 60 min, and the gradient elution conditions were shown in Table 1. The injection volume was 5 μL, and the flow rate was set at 300 μL / min.

[0021] Mass spectrometry analysis was performed in positive ion mode, using a two-stage tandem mass spectrometry with a resolution of 120,000 and a full MS scan from 60 to 2000 m / z. The specific mass spectrometry parameter settings are shown in Table 2.

[0022] 2) Mass spectrometry data analysis The raw files obtained from UPLC-MS / MS analysis were converted into MGF format mass spectrometry general files using MM File Conversion software. The protein data in the uniport database (http: / / www.uniprot.org / taxonomy / 8139) were retrieved using the Mascot mass spectrometry data online analysis platform (http: / / www.matrixscience.com / ) to analyze the mass spectrometry information in the MGF files. The specific search parameters were as follows: Fixed modifications (Carbamidomethyl, C), Variable Modifications (Oxidation, M), Enzyme (none), Maximum Missed Cleavages (1), Peptide Mass Tolerance (1.2 Da), Fragment Mass Tolerance (0.6 Da), Mass values ​​(Monoisotopic), Peptide charge (1+, 2+ and 3+), and Significance threshold (0.05).

[0023] 3) Soy peptides were identified by UPLC-ESI-TOF-MS / MS. Mass spectrometry analysis identified 757 peptide sequences with a molecular weight distribution range of 200–1000 Da. Bioinformatics prediction was performed for each soy peptide sequence. Based on the bioinformatics analysis results, non-toxic and highly active soy peptides were selected for chemical synthesis and subsequently assayed for in vitro and in vivo antioxidant and anti-aging activities.

[0024] (5) Prediction and analysis of biological activity of soybean peptides: 1) Prediction of potential biological activity of peptides The potential biological activities of the peptide sequences were analyzed using the PeptideRanker online platform (http: / / distilldeep.ucd). Peptides were ranked based on their predicted probability of biological activity. The PeptideRanker prediction model was pre-set at a threshold of 0.5; peptides with a threshold greater than 0.5 were considered biologically active, with higher thresholds indicating higher activity. The predicted scores for the screened soybean peptides VLLPGP, PAGLYP, and FLVLPA were 0.5, 0.53, and 0.56, respectively (see Table 3). Bioinformatics prediction analysis indicated that VLLPGP, PAGLYP, and FLVLPA had high potential biological activities.

[0025] Table 3 Mass spectrometry analysis and biological activity prediction analysis results of soybean peptides

[0026] 2) Prediction of potential toxicity of soybean peptides The ToxinPrep (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php) platform was used to predict the potential toxicity of structurally elucidated soybean peptides based on the SVM (Swiss-Port) algorithm.

[0027] Toxicity prediction analysis showed that VLLPGP, PAGLYP, and FLVLPA were all non-toxic peptides.

[0028] 3) Prediction of antioxidant activity of soybean peptides The free radical scavenging (FRS) and metal ion chelating (CHEL) abilities of identified soybean peptide sequences with a PeptideRanker greater than 0.5 and no toxicity were predicted using the AnOxPePred platform. Peptide sequences with FRS values ​​greater than 0.5 or CHEL values ​​greater than 0.25 were screened. The prediction results are shown in Table 4.

[0029] Table 4 Prediction analysis results of antioxidant activity of soybean peptides

[0030] 4) Bioinformatics analysis identified three soybean peptides with amino acid sequences: VLLPGP, PAGLYP, and FLVLPA (SEQ ID NOs. 1, 2, and 3). These peptides were synthesized using Fmoc solid-phase synthesis technology. The peptides were synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. The purity of the synthesized peptides was >95%.

[0031] Test Example 1 In vitro antioxidant activity assay of SPAP2: The antioxidant activity of SPAP2 was determined by ABTS free radical scavenging test. 7.4mmol / L ABTS stock solution and 2.6mmol / L K2S2O8 stock solution were prepared respectively. 0.2mL of 7.4mmol / L ABTS stock solution and 0.2mL of 2.6mmol / L K2S2O8 stock solution were measured and mixed. The mixture was allowed to react at room temperature in the dark for 12 hours to prepare ABTS. + Free radical stock solution. Dilute ABTS in PBS (5.0 mM, pH 7.4). + The mother liquor is converted to an A734 value of 1.000-1.099 to obtain ABTS. + Free radical working solution. Add 200uL diluted ABTS +The free radical working solution was added to 20 μL of the SPAP2 solution obtained in Example 1. The reaction was carried out at room temperature in the dark for 10 minutes, and the absorbance was measured at 734 nm. Three replicates were performed for each group, and the average value was calculated.

[0032] Calculate the ABTS free radical scavenging rate using the following formula: ABTS free radical scavenging rate (%) = [(Ac - As) / Ac] × 100%. Where: As is the absorbance of the sample group, i.e., the absorbance of the sample and ABTS free radicals; Ac is the absorbance of the control group, i.e., the absorbance of the phosphate buffer and ABTS free radicals.

[0033] The ABTS free radical scavenging rate of SPAP2 was determined by ABTS method (e.g. Figure 1 As shown). After calculation, the ABTS free radical scavenging rate IC of SPAP2 50 The value was 0.591±0.028 mg / mL.

[0034] Test Example 2 Animal experiments evaluating the anti-aging effects of SPAP2 in vivo: Synchronized L4-stage Caenorhabditis elegans adults were randomly selected and plated on NGM plates containing OP50 bacterial lawns, with 30 worms per group. Experimental and control groups were established. Experimental groups were treated with 20.0 μL of 0.25 mg / mL (low-dose group), 0.50 mg / mL (medium-dose group), and 1.00 mg / mL (high-dose group) SPAP2 solution sterilized with a 0.22 μm filter, along with 20.0 μL of 5-fluorouracil (75 μg / mL). Control groups were treated with M9 buffer instead of sample and incubated at 20°C. The day of transfer was designated as day 0. At the same time each day, C. elegans were transferred to new NGM plates containing OP50 bacterial lawns. Survival and mortality were observed and recorded daily. The worms' response to external stimuli was used as the criterion for mortality. Observation was continued until all worms had died. Mean and maximum lifespans were calculated.

[0035] Survival curves of nematodes treated with different concentrations of SPAP2 Figure 2 The average lifespan of nematodes in the control group was 18.29±0.08 d, while the lifespans of nematodes treated with low, medium, and high concentrations of SPAP2 were 20.36±0.49 d, 21.59±0.22 d, and 22.32±0.19 d, respectively. This extended the average lifespan of nematodes by 11.32±2.90%, 18.04±1.09%, and 22.03±1.37%, respectively. Medium and high concentrations of SPAP2 significantly increased the average lifespan of nematodes ( p <0.05).

[0036] Test Example 3 Determination of the antioxidant activity of soybean peptides in vitro The ABTS free radical scavenging rate test method and steps are the same as those in Test Example 1.

[0037] After calculation, the ABTS free radical scavenging rates IC 50 The values ​​are 0.65mM, 1.74mM, and 0.79mM respectively.

[0038] Test Example 4 Determination of the antioxidant and anti-aging activities of soybean peptides in vivo (1) Effect of soybean peptide on the lifespan of nematodes under oxidative stress: L4 synchronized Caenorhabditis elegans were randomly selected and inoculated on NGM plates containing OP50 bacteria, with 30 nematodes in each group. Experimental and control groups were set up. The experimental group was added with 20 μL of 1 mg / mL peptide solution and 20 μL of 75 μg / mL 5-fluorouracil that had been sterilized with a 0.22 μm filter membrane, while the control group was replaced with M9 buffer instead of peptide solution. After incubation at 20°C for 24 h, the Caenorhabditis elegans on the plate were rinsed with M9 buffer, collected into a 2 mL centrifuge tube, rinsed with M9 buffer, and 500 μL of 10 mM H2O2 was added for 1 h. The survival of the nematodes was recorded under a microscope until all the nematodes in each group died. The survival rate of the nematodes was calculated.

[0039] The results of oxidative stress survival rate determination showed that the average lifespan of C. elegans in the control group was 6.95±0.95h; while the average lifespan of C. elegans treated with peptides was significantly prolonged. VLLPGP, PAGLYP, and FLVLPA extended the average lifespan of C. elegans to 9.60±0.18 h, 8.00±0.73 h, and 8.87±0.82 h, respectively, and the average lifespan increased by 38.2%, 15.12%, and 27.58%, respectively.

[0040] (2) Effect of soybean peptide on antioxidant indicators in nematodes under oxidative stress: Synchronized L4-stage Caenorhabditis elegans were randomly selected and inoculated into NGM plates containing OP50 bacteria, with 30 worms per group. Experimental and control groups were set up. 20 μL of 1 mg / mL peptide solution sterilized with a 0.22 μm filter and 20 μL of 75 μg / mL 5-fluorouracil were added to the experimental group. 20 μL of M9 buffer and 20 μL of 75 μg / mL 5-fluorouracil were added to the control group. The cells were cultured at 20°C for 24 h. The nematodes were collected in a centrifuge tube, rinsed with M9 buffer, and transferred to a new centrifuge tube. 10 mM H2O2 was added for 1 h, and then washed with physiological saline. The nematode cells were broken into a homogenate with a final concentration of 10%, centrifuged, and the supernatant was collected. According to the kit instructions, superoxide dismutase (SOD) activity and protein content were determined, and the antioxidant enzyme activity was calibrated with protein content.

[0041] The peptide's oxidative regulatory effect on C. elegans was evaluated by measuring superoxide dismutase (SOD) levels in C. elegans under oxidative stress. The results are shown in Table 5. Compared with the control group, peptide-treated C. elegans exhibited significantly increased SOD activity and decreased ROS levels. These results suggest that the peptide exerts its antioxidant effect by increasing SOD activity and reducing ROS levels in C. elegans.

[0042] Table 5 Determination results of in vivo antioxidant indexes in oxidative stress Caenorhabditis elegans treated with peptides

[0043] (3) Determination of the anti-aging activity of soybean peptides in nematodes: Synchronized L4-stage Caenorhabditis elegans adults were randomly selected and inoculated into NGM plates containing OP50 bacterial moss, with 50 worms in each group. Experimental and control groups were set up. 20 μL of 1 mg / mL peptide solution and 20 μL of 75 μg / mL 5-fluorouracil sterilized with a 0.22 μm filter were added to the experimental group, while M9 buffer was used instead of peptide solution in the control group. The culture was carried out at 20°C. The day of transfer of Caenorhabditis elegans was recorded as day 0. At the same time every day, Caenorhabditis elegans were transferred to a new NGM plate containing OP50 bacterial moss. The number of Caenorhabditis elegans deaths was observed and recorded every day. Whether Caenorhabditis elegans responded to external stimuli was used as the basis for judging their death. The experiment was terminated when all Caenorhabditis elegans died. The average lifespan of the nematodes was calculated.

[0044] To evaluate the effects of peptides on the lifespan of C. elegans, C. elegans were treated with soybean peptides at a concentration of 1 mg / mL. The average lifespan of C. elegans in the control group was 18.29±0.08 d, while the average lifespan of C. elegans treated with VLLPGP, PAGLYP, and FLVLPA was 26.34±0.21 d, 22.58±0.03 d, and 25.65±0.16 d, respectively, which were 44.03%, 23.46%, and 40.22% longer than those in the control group, respectively. Biostatistical analysis showed that there were significant differences ( p <0.05). Anti-aging test results showed that all three peptides could significantly extend the lifespan of Caenorhabditis elegans.

[0045] The above in vivo and in vitro analysis results show that soybean antioxidant peptides VLLPGP, PAGLYP, and FLVLPA have antioxidant and anti-aging activities and can be used to develop antioxidant and anti-aging related cosmetics and medicines.

[0046] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A soybean active peptide with antioxidant and anti-aging activities, characterized in that: The active peptide is selected from SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; the amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 are Val-Leu-Leu-Pro-Gly-Pro, Pro-Ala-Gly-Leu-Tyr-Pro and Phe-Leu-Val-Leu-Pro-Ala, respectively.

2. The method for preparing the soybean active peptide according to claim 1, wherein The preparation method comprises the following steps: (1) Using soybean protein as raw material, add distilled water, mix well, sterilize with high-pressure steam, and cool to room temperature to obtain a sterilized soybean protein solution; (2) Probiotic liquid fermentation of soybean protein to prepare soybean active peptides: inoculate Bacillus natto into the sterilized soybean protein solution obtained in step (1), shake culture at 37°C for 24 h, and shake at a speed of 100-200 rpm; then inoculate Bacillus coagulans, shake culture at 47°C for 42 h, and shake at a speed of 100-200 rpm; then inoculate Lactobacillus rhamnosus, shake culture at 37°C for 36 h, and shake at a speed of 100-200 rpm; obtain fermentation liquid; (3) The fermentation broth was ultrafiltered using an ultrafiltration centrifuge tube with a molecular weight cutoff of 1 kDa. The filtrate was centrifuged at 10,000-12,000 rpm for 15 min at 4°C, and the filtrate was collected as the soybean peptide solution, which was named SPAP2. (4) Filter SPAP2 to remove particles, and perform mass spectrometry identification using ultra-high performance liquid chromatography-tandem mass spectrometry to obtain the soybean peptide sequence; (5) Bioinformatics prediction of each soybean peptide sequence: 1) Prediction of potential biological activity of peptides: Use PeptideRanker to predict and analyze the biological activity of peptides, and screen peptide sequences with PeptideRanker prediction values ​​greater than 0.5 for subsequent analysis; 2) Peptide toxicity prediction analysis: ToxinPrep, based on the SVM algorithm, was used to perform potential toxicity prediction analysis on peptide sequences with a PeptideRanker prediction value greater than 0.5 in step 1); 3) Prediction of antioxidant activity of soybean peptides: For soybean peptide sequences with PeptideRanker > 0.5 and no toxicity in step 2), AnOxPePred was used to predict their free radical scavenging ability and metal ion chelating ability, and peptide sequences with FRS values ​​> 0.5 or CHEL values ​​> 0.25 were screened to obtain soybean active peptides.

3. The preparation method according to claim 2, characterized in that The mass ratio of soy protein to distilled water in step (1) is 1:

20.

4. The preparation method according to claim 2, characterized in that In step (1), high pressure steam sterilization is performed at 121° C. for 15 minutes.

5. The preparation method according to claim 2, characterized in that In the step (2), the inoculation amount of Bacillus natto, Bacillus coagulans and Lactobacillus rhamnosus is 2% of the mass of the sterilized soy protein solution.

6. The preparation method according to claim 2, wherein In the step (4), SPAP2 is filtered using a 0.22 μm water syringe filter.

7. The preparation method according to claim 2, wherein In step (4), the mobile phase A of the liquid chromatography is an aqueous solution containing 0.1% formic acid, the mobile phase B is an acetonitrile solution containing 0.1% formic acid, the elution time is 60 min, the injection volume is 5 μL, the flow rate is set to 300 μL / min, and the gradient elution conditions are: 0-5 min, 5% B; 5-45 min, 5% B; 45-50 min, 50% B; 50-55 min, 90% B; 55-60 min, 90% B; >60min, 5%B.

8. The preparation method according to claim 2, wherein In the step (4), mass spectrometry identification was performed in positive ion mode, with a secondary tandem mass spectrometry analysis, a resolution of 120,000, a full MS scan of 60-2000 m / z, a capillary voltage of 3500 V, a cone voltage of 20 V, an ion source temperature of 100°C, a desolvation temperature of 400°C, a desolvation gas flow rate of 700 L / h, a cone gas flow rate of 50 L / h, a collision voltage of 6-25 eV, and a mass spectrum acquisition speed of 2-4 frames / second.

9. Use of the soybean active peptide according to claim 1 in the preparation of antioxidant, anti-aging drugs or cosmetics.

10. Use of the soybean active peptide obtained by the preparation method according to any one of claims 2 to 8 in the preparation of antioxidant and anti-aging drugs or cosmetics.

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