Soybean bioactive peptides with antioxidant and anti-aging activities, their preparation methods and applications

Abstract

This invention relates to soybean bioactive peptides with antioxidant and anti-aging activities, their preparation methods, and applications, belonging to the field of bioactive peptide and protein technology. The bioactive peptides are 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 VLLPGP, PAGLYP, and FLVLPA, respectively. The soybean bioactive peptides of this invention have been experimentally verified to possess antioxidant and anti-aging activities, and are safe and free of toxic side effects. They can be used as functional ingredients in antioxidant and anti-aging cosmetics, pharmaceuticals, etc., showing good potential and application prospects.

Description

Technical Field

[0001] This invention relates to soybean bioactive peptides with antioxidant and anti-aging activities, their preparation methods and applications, and belongs to the field of bioactive peptide protein technology. Background Technology

[0002] Peptides are fragments composed of 2-20 amino acid residues, produced after the degradation of proteins. Based on their biological activities, peptides are classified into antioxidant peptides, anti-aging peptides, anti-inflammatory peptides, antimicrobial peptides, blood sugar-lowering peptides, blood pressure-lowering peptides, lipid-lowering peptides, anticancer peptides, and immune peptides, among others. Antioxidant peptides refer to polypeptides with antioxidant activity and can be used in functional foods, pharmaceuticals, cosmetics, etc., showing broad application prospects.

[0003] The production and accumulation of free radicals in the human body can lead 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, thus preventing and treating diseases and delaying aging. Butylated hydroxyanisole, tert-butylhydroquinone, butylated hydroxytoluene, and propyl gallate are commonly used chemically synthesized antioxidants. However, these chemicals pose certain safety risks. Antioxidant peptides, derived from food proteins, have gained attention both domestically and internationally due to 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, rich in essential amino acids. The main components of soybean protein are β-conglycinin and glycinin, which together account for 80%-90% of the total soybean protein. Soy protein degradation produces soybean bioactive peptides. The main methods for preparing soybean bioactive peptides include enzymatic hydrolysis, microbial fermentation, and chemical processing. Microbial fermentation, which utilizes specific microorganisms and their produced proteases to hydrolyze soybean protein, offers advantages such as low production cost, mild reaction conditions, simple 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 soybean fermentation include bacteria such as *Lactococcus*, *Lactobacillus*, *Streptococcus thermophilus*, and *Bacillus subtilis*, as well as fungi such as *Saccharomyces* and *Aspergillus niger*. The sequences and activities of soybean peptides obtained from fermentation by different microorganisms vary.

[0005] To date, several bioactive soybean peptides have been screened from soybean protein degradation products, including soybean hypoglycemic peptides, soybean immune peptides, soybean anti-inflammatory peptides, soybean antihypertensive peptides, soybean lipid-lowering peptides, soybean anticancer peptides, and soybean antioxidant peptides. Reported soybean antioxidant peptide sequences include NPESQQGSPRV, PGTAVFK, IKAFKEATKVDKVVVLWTA, VNPESQQGSPR, and LH. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides soybean bioactive peptides with antioxidant and anti-aging activities, their preparation methods, and applications. The soybean bioactive peptides obtained by this invention have the advantages of being safe and having no toxic side effects.

[0007] The technical solution of the present invention is as follows:

[0008] Soybean bioactive peptides with antioxidant and anti-aging activities, wherein the bioactive peptides are 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 respectively: Val-Leu-Leu-Pro-Gly-Pro (VLLPGP), Pro-Ala-Gly-Leu-Tyr-Pro (PAGLYP) and Phe-Leu-Val-Leu-Pro-Ala (FLVLPA).

[0009] The preparation method of the above-mentioned soybean bioactive peptides includes the following steps:

[0010] (1) Using soybean protein as raw material, add distilled water at a material-to-liquid ratio of 1:20 (w / w), mix well, sterilize by high pressure steam at 121℃ for 15 min, and cool to room temperature to obtain sterilized soybean protein solution;

[0011] (2) Preparation of soybean bioactive peptides by probiotic liquid fermentation of soybean protein: Bacillus natto was inoculated into the sterilized soybean protein solution obtained in step (1) and cultured at 37°C with shaking for 24 h at a shaking speed of 100-200 rpm; then Bacillus coagulans was inoculated and cultured at 47°C with shaking for 42 h at a shaking speed of 100-200 rpm; then Lactobacillus rhamnosus was inoculated and cultured at 37°C with shaking for 36 h at a shaking speed of 100-200 rpm; the fermentation broth was obtained.

[0012] Preferably, in step (2), the inoculation amount of Bacillus natto, Bacillus coagulans and Lactobacillus rhamnosus is 2% of the mass of the sterilized soybean protein solution.

[0013] (3) The fermentation broth was separated by ultrafiltration using an ultrafiltration centrifuge tube with a molecular weight cutoff of 1 kDa. The mixture was centrifuged at 10,000-12,000 rpm for 15 min at 4°C. The filtrate was collected and was the soybean peptide solution, named SPAP2.

[0014] (4) SPAP2 particles were removed by filtering with a 0.22 μm aqueous syringe filter and identified by mass spectrometry using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-TOF-MS / MS) to obtain the soybean peptide sequence;

[0015] Preferably, the mobile phase A of the 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.

[0016] Table 1 Elution conditions for ultra-high performance liquid chromatography

[0017]

[0018] Preferably, the mass spectrometry analysis adopts positive ion mode, two-stage tandem mass spectrometry analysis, with a resolution of 120,000, and performs full MS scans of 60-2000 m / z. The tandem mass spectrometry analysis conditions are shown in Table 2.

[0019] Table 2 Tandem Mass Spectrometry Analysis Conditions

[0020]

[0021] (5) Bioinformatics prediction of each soybean peptide sequence:

[0022] 1) Prediction of potential biological activity of peptides: PeptideRanker was used to predict and analyze the biological activity of peptides, and peptide sequences with a PeptideRanker prediction value > 0.5 were screened for further analysis.

[0023] 2) Peptide toxicity prediction analysis: ToxinPrep, based on the SVM algorithm, was used to perform potential toxicity prediction analysis on peptide sequences with PeptideRanker prediction values ​​> 0.5 in step 1).

[0024] 3) Antioxidant prediction 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 (FRS) and metal ion chelating ability (CHEL). Peptide sequences with FRS value > 0.5 or CHEL value > 0.25 were screened to obtain soybean bioactive peptides VLLPGP, PAGLYP, and FLVLPA.

[0025] The present invention also includes the application of the above-mentioned soybean bioactive peptides in the preparation of antioxidant and anti-aging drugs or cosmetics.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] This invention provides the first screening of three novel peptides, VLLPGP, PAGLYP, and FLVLPA, from soybean. Bioinformatics prediction results indicate that VLLPGP, PAGLYP, and FLVLPA have no toxic side effects and high bioactivity. In vitro antioxidant experiments show that VLLPGP, PAGLYP, and FLVLPA have an IC50 scavenging rate of ABTS free radicals. 50 The values ​​were 0.65 mM, 1.74 mM, and 0.79 mM, respectively. The in vivo antioxidant and anti-aging activities of soybean peptides were evaluated in the model organism *C. elegans*. Oxidative stress assays showed that 1 mg / mL solutions of VLLPGP, PAGLYP, and FLVLPA increased the average lifespan of *C. elegans* under oxidative stress by 38.2%, 15.12%, and 27.58%, respectively. Anti-aging assays showed that 1 mg / mL peptide solutions of VLLPGP, PAGLYP, and FLVLPA prolonged the average lifespan of *C. elegans* by 44.03%, 23.46%, and 40.22%, respectively, significantly different from the control group. In vivo antioxidant assays confirmed that VLLPGP, PAGLYP, and FLVLPA could increase the activity of SOD and reduce ROS content in vivo, thereby exerting antioxidant and anti-aging effects. In summary, both in vivo and in vitro experimental results confirm that the novel peptides VLLPGP, PAGLYP, and FLVLPA possess antioxidant and anti-aging activities. Therefore, novel antioxidant and anti-aging soybean peptides VLLPGP, PAGLYP, and FLVLPA can be used as functional ingredients in antioxidant and anti-aging cosmetics, pharmaceuticals, etc., and have good application prospects. Attached Figure Description

[0028] Figure 1 The results of the ABTS free radical scavenging rate determination of SPAP2 obtained in Example 1;

[0029] Figure 2 Survival curves of nematodes treated with different concentrations of SPAP2. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0031] The Bacillus subtilis, Bacillus coagulans, and Lactobacillus rhamnosus used in the embodiments of this invention were all purchased from microbial culture collection centers. Bacillus subtilis (CGMCC 4731) was purchased from the China General Microbiological Culture Collection Center; Bacillus coagulans (CCTCC AB 2018147) was purchased from the China Center for Type Culture Collection; and Lactobacillus rhamnosus (CICC21006) was purchased from the China Industrial Microbiological Culture Collection Center.

[0032] Example 1

[0033] Soybean bioactive peptides with antioxidant and anti-aging activities, wherein the bioactive peptides are selected from SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0034] The preparation method of the above-mentioned soybean bioactive peptides includes the following steps:

[0035] (1) Using soybean protein as raw material, add distilled water at a material-to-liquid ratio of 1:20 (w / w), mix well, sterilize by high pressure steam at 121℃ for 15 minutes, and wait for the soybean protein solution temperature to drop to room temperature.

[0036] (2) Prepare soybean active peptides by inoculating probiotics into the sterilized soybean protein solution: First, inoculate with Bacillus natto (inoculation amount of 2%, w / w), and culture at 37℃ with shaking for 24 h at a shaking speed of 180 rpm; then inoculate with Bacillus coagulans (inoculation amount of 2%, w / w), and culture at 47℃ with shaking for 42 h at a shaking speed of 180 rpm; then inoculate with Lactobacillus rhamnosus (inoculation amount of 2%, w / w), and culture at 37℃ with shaking for 36 h at a shaking speed of 180 rpm;

[0037] (3) The fermentation broth was separated by ultrafiltration using an ultrafiltration centrifuge tube with a molecular weight cutoff of 1 kDa. The mixture was centrifuged at 10,000 rpm for 15 min at 4°C. The ultrafiltration fraction with a molecular weight cutoff of <1 kDa was collected, which was the soybean peptide solution and named SPAP2.

[0038] (4) Mass spectrometry identification of SPAP2:

[0039] 1) SPAP2 particles were removed by filtering with a 0.22 μm aqueous syringe filter, and then identified by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-TOF-MS / MS). The mobile phase A of the UPLC was an aqueous solution (containing 0.1% formic acid), and the mobile phase B was an acetonitrile solution (containing 0.1% formic acid). The elution time was 60 min, and the gradient elution conditions are shown in Table 1. The injection volume was 5 μL, and the flow rate was set to 300 μL / min.

[0040] Mass spectrometry analysis was performed in positive ion mode, using two-stage tandem mass spectrometry with a resolution of 120,000, and a full MS scan of 60-2000 m / z. Specific mass spectrometry parameter settings are shown in Table 2.

[0041] 2) Mass spectrometry data analysis

[0042] The raw files obtained from UPLC-MS / MS analysis were converted into MGF format mass spectrometry universal files using MM File Conversion software. Protein data in the Uniport database (http: / / www.uniprot.org / taxonomy / 8139) were retrieved using the Mascot online mass spectrometry data analysis platform (http: / / www.matrixscience.com / ). The mass spectrometry information in the MGF files was analyzed. Specific search parameters were: 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).

[0043] 3) Soybean peptides were identified by UPLC-ESI-TOF-MS / MS. Mass spectrometry analysis identified 757 peptide sequences, with molecular weights ranging from 200 to 1000 Da. Bioinformatics prediction was performed on each soybean peptide sequence. Based on the bioinformatics analysis results, non-toxic and highly active soybean peptides were selected for chemical synthesis, followed by in vivo and in vitro assays of their antioxidant and anti-aging activities.

[0044] (5) Predictive analysis of the biological activity of soybean peptides:

[0045] 1) Prediction of the potential biological activity of peptides

[0046] The potential biological activity of the obtained peptide sequences was analyzed using the PeptideRanker online platform (http: / / distilldeep.ucd). Peptides were ranked according to their predicted biological activity probabilities. The preset threshold for the PeptideRanker prediction model was 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 results indicate that VLLPGP, PAGLYP, and FLVLPA have high potential biological activity.

[0047] Table 3. Mass spectrometry analysis and biological activity prediction results of soybean peptides

[0048]

[0049] 2) Prediction of the potential toxicity of soybean peptides

[0050] The potential toxicity of soybean peptides with resolved structures was predicted using the ToxinPrep platform (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php) based on the SVM (Swiss-Port) algorithm.

[0051] According to toxicity prediction analysis, VLLPGP, PAGLYP, and FLVLPA are all non-toxic peptides.

[0052] 3) Prediction of the antioxidant properties of soybean peptides

[0053] Soybean peptide sequences with a PeptideRanker > 0.5 and no toxicity were used to predict their free radical scavenging (FRS) and metal ion chelating (CHEL) capabilities using the AnOxPePred platform. Peptide sequences with an FRS value > 0.5 or a CHEL value > 0.25 were then screened. The prediction results are shown in Table 4.

[0054] Table 4. Predictive analysis results of antioxidant activity of soybean peptides

[0055]

[0056] 4) Bioinformatics analysis identified three soybean peptides with amino acid sequences of VLLPGP, PAGLYP, and FLVLPA (SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively). These peptides were synthesized using Fmoc amino acid solid-phase synthesis technology. The peptides were synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. The purity of the synthesized peptides was >95%.

[0057] Experimental Example 1

[0058] In vitro antioxidant activity assay of SPAP2:

[0059] The antioxidant activity of SPAP2 was determined using the ABTS free radical scavenging assay. 7.4 mmol / L ABTS stock solutions and 2.6 mmol / L K₂S₂O₈ stock solutions were prepared. 0.2 mL of each solution was mixed and reacted at room temperature in the dark for 12 hours to obtain ABTS. + Free radical stock solution. Dilute ABTS with PBS (5.0 mM, pH 7.4). + The mother liquor was diluted to an A734 value of 1.000-1.099 to obtain ABTS. + Free radical working solution. 200 μL of diluted ABTS + The free radical working solution was added to 20 μL of the SPAP2 solution obtained in Example 1, and the reaction was carried out at room temperature in the dark for 10 min. The absorbance was measured at 734 nm. Each group was repeated in 3 replicates, and the average value was taken.

[0060] The ABTS radical scavenging rate is calculated using the following formula: ABTS radical scavenging rate (%) = [(Ac-As) / Ac] × 100%. Where: As is the absorbance of the sample group, which is the absorbance of the sample and ABTS radicals; Ac is the absorbance of the control group, which is the absorbance of the phosphate buffer and ABTS radicals.

[0061] ABTS method for determining the ABTS radical scavenging rate of SPAP2 (e.g.) Figure 1 (As shown). The calculated ABTS radical scavenging rate IC of SPAP2 is... 50 The value was 0.591 ± 0.028 mg / mL.

[0062] Experimental Example 2

[0063] Animal studies evaluating the in vivo anti-aging effects of SPAP2:

[0064] Synchronized L4-stage adult *C. elegans* were randomly selected and inoculated into NGM plates containing OP50 bacterial culture, with 30 worms per group. Experimental and control groups were established. The experimental groups received 20.0 μL of SPAP2 solution (sterilized with a 0.22 μm filter at concentrations of 0.25 mg / mL (low-dose), 0.50 mg / mL (medium-dose), and 1.00 mg / mL (high-dose), respectively), and 20.0 μL of 5-fluorouracil (75 μg / mL). The control group received M9 buffer instead of the sample. All samples were incubated at 20°C. The day of transfer of *C. elegans* was designated as day 0. At the same time each day, *C. elegans* were transferred to new NGM plates containing OP50 bacterial culture. The number of surviving and dead *C. elegans* worms was observed and recorded daily, with responsiveness to external stimuli used to determine mortality. The observation continued until all *C. elegans* worms had died. The average and maximum lifespan of the nematodes were calculated.

[0065] Survival curves of nematodes treated with different concentrations of SPAP2 are shown below. Figure 2 As shown. The average lifespan of nematodes in the control group was 18.29±0.08 days. The lifespans of nematodes treated with low, medium, and high concentrations of SPAP2 were 20.36±0.49 days, 21.59±0.22 days, and 22.32±0.19 days, respectively, extending 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).

[0066] Experimental Example 3

[0067] In vitro antioxidant activity assay of soybean peptides

[0068] The test method and procedure for ABTS free radical scavenging rate are the same as those in Test Example 1.

[0069] Calculations showed that VLLPGP, PAGLYP, and FLVLPA had an ABTS radical scavenging rate IC50 of [missing value]. 50 The values ​​were 0.65 mM, 1.74 mM, and 0.79 mM, respectively.

[0070] Test Example 4

[0071] In vivo antioxidant and anti-aging activity assay of soybean peptides

[0072] (1) Effect of soybean peptides on the lifespan of nematodes under oxidative stress: L4-stage synchronized *C. elegans* were randomly selected and inoculated onto NGM plates containing OP50 bacterial culture, with 30 nematodes per group. An experimental group and a control group were established. The experimental group received 20 μL of a 1 mg / mL peptide solution and 20 μL of 75 μg / mL 5-fluorouracil, which had been pre-sterilized using a 0.22 μm filter. The control group received M9 buffer instead of the peptide solution. After incubation at 20℃ for 24 h, the *C. elegans* on the plates were rinsed with M9 buffer, and the nematodes were collected into 2 mL centrifuge tubes. The tubes were rinsed thoroughly with M9 buffer, and 500 μL of 10 mM H2O2 was added. After 1 h of incubation, the survival of the nematodes was recorded under a microscope until all nematodes in each group died. The survival rate of the nematodes was then calculated.

[0073] The survival rate determination results under oxidative stress showed that the average lifespan of *C. elegans* in the control group was 6.95 ± 0.95 h; 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, representing increases of 38.2%, 15.12%, and 27.58%.

[0074] (2) Effect of soybean peptides on in vivo antioxidant indices of nematodes under oxidative stress: L4-stage synchronized Caenorhabditis elegans adults were randomly selected and inoculated into NGM plates containing OP50 bacterial culture, with 30 nematodes per group. An experimental group and a control group were established. The experimental group received 20 μL of a 1 mg / mL peptide solution sterilized by a 0.22 μm filter and 20 μL of 75 μg / mL 5-fluorouracil. The control group received 20 μL of M9 buffer and 20 μL of 75 μg / mL 5-fluorouracil. The mixture was incubated at 20℃ for 24 h. The nematodes were collected in centrifuge tubes, rinsed thoroughly with M9 buffer, transferred to new centrifuge tubes, and treated with 10 mM H2O2 for 1 h. Afterward, the tubes were washed with physiological saline, the nematode cells were broken, and a homogenate with a final concentration of 10% was prepared. The homogenate was centrifuged, and the supernatant was collected. According to the kit instructions, the activity and protein content of superoxide dismutase (SOD) were measured, and the activity of the antioxidant enzyme was determined by the protein content.

[0075] The antioxidant regulatory effect of peptides on *C. elegans* was evaluated by measuring the superoxide dismutase (SOD) content in *C. elegans* under oxidative stress. The results are shown in Table 5. Compared with the control group, the SOD activity in *C. elegans* treated with peptides under oxidative stress was significantly increased, while the ROS content was decreased. The experimental results indicate that peptides exert their antioxidant regulatory effect by increasing SOD activity and reducing ROS content in *C. elegans*.

[0076] Table 5. Results of in vivo antioxidant index determination of Caenorhabditis elegans under oxidative stress treated with peptides.

[0077]

[0078] (3) Assay of the anti-aging activity of soybean peptides against nematodes: Synchronized L4-stage adult *C. elegans* were randomly selected and inoculated into NGM plates containing OP50 bacterial culture, 50 nematodes per group. An experimental group and a control group were established. The experimental group received 20 μL of a 1 mg / mL peptide solution sterilized with a 0.22 μm filter and 20 μL of 75 μg / mL 5-fluorouracil. The control group received M9 buffer instead of the peptide solution. The nematodes were incubated at 20℃. The day of transfer of *C. elegans* was recorded as day 0. Each day at the same time, *C. elegans* were transferred to new NGM plates containing OP50 bacterial culture. The number of dead *C. elegans* nematodes was observed and recorded daily. The response of *C. elegans* to external stimuli was used as the basis for determining mortality. The experiment continued until all *C. elegans* nematodes died. The average lifespan of the nematodes was calculated.

[0079] To evaluate the effect 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. The average lifespans of *C. elegans* treated with VLLPGP, PAGLYP, and FLVLPA were 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 all differences were statistically significant. p <0.05). Anti-aging test results showed that all three peptides significantly prolonged the lifespan of *C. elegans*.

[0080] The above in vivo and in vitro analysis results indicate that soybean antioxidant peptides VLLPGP, PAGLYP, and FLVLPA possess antioxidant and anti-aging activities, and can be used to develop antioxidant and anti-aging related cosmetics and pharmaceuticals.

[0081] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A soybean bioactive 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 soybean bioactive peptides as described in claim 1, characterized in that, The preparation method includes the following steps: (1) Using soybean protein as raw material, add distilled water, mix well, sterilize by high pressure steam, and cool to room temperature; to obtain sterilized soybean protein solution; (2) Preparation of soybean bioactive peptides by probiotic liquid fermentation of soybean protein: Bacillus natto was inoculated into the sterilized soybean protein solution obtained in step (1), and cultured at 37°C with shaking for 24 h at a shaking speed of 100-200 rpm; then Bacillus coagulans was inoculated, and cultured at 47°C with shaking for 42 h at a shaking speed of 100-200 rpm; then Lactobacillus rhamnosus was inoculated, and cultured at 37°C with shaking for 36 h at a shaking speed of 100-200 rpm; fermentation broth was obtained; the Bacillus natto preservation number is: CGMCC 4731; the Bacillus coagulans preservation number is: CCTCC AB 2018147; the Lactobacillus rhamnosus preservation number is: CICC21006; the inoculation amount of Bacillus natto, Bacillus coagulans and Lactobacillus rhamnosus is 2% of the mass of the sterilized soybean protein solution; (3) The fermentation broth was separated by ultrafiltration using an ultrafiltration centrifuge tube with a molecular weight cutoff of 1 kDa. The mixture was centrifuged at 10,000-12,000 rpm for 15 min at 4°C. The filtrate was collected and was the soybean peptide solution, named SPAP2. (4) Filter SPAP2 to remove particles, and use ultra-high performance liquid chromatography-tandem mass spectrometry to identify the soybean peptide sequence. (5) Bioinformatics prediction of each soybean peptide sequence: 1) Prediction of potential biological activity of peptides: PeptideRanker was used to predict and analyze the biological activity of peptides, and peptide sequences with a PeptideRanker prediction value > 0.5 were screened for further analysis. 2) Peptide toxicity prediction analysis: ToxinPrep, based on the SVM algorithm, was used to perform potential toxicity prediction analysis on peptide sequences with PeptideRanker prediction values ​​> 0.5 in step 1). 3) Antioxidant prediction 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. Peptide sequences with FRS value > 0.5 or CHEL value > 0.25 were screened to obtain soybean bioactive peptides.

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

20.

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

5. The preparation method according to claim 2, characterized in that, In step (4), SPAP2 is filtered using a 0.22 μm water-based needle filter.

6. The preparation method according to claim 2, characterized in that, In step (4), the mobile phase A of the liquid chromatography is an aqueous solution containing 0.1% formic acid, and 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.

7. The preparation method according to claim 2, characterized in that, In step (4), the mass spectrometry identification adopts positive ion mode, two-stage tandem mass spectrometry analysis, with a resolution of 120,000, and performs full MS scans of 60-2000 m / z. The capillary voltage is 3500V, the cone voltage is 20V, the ion source temperature is 100℃, the desolventizing temperature is 400℃, the desolventizing gas flow rate is 700 L / h, the cone gas flow rate is 50L / h, the collision voltage is 6-25 eV, and the mass spectrum acquisition speed is 2-4 frames / second.

8. The application of the soybean bioactive peptide as described in claim 1 in the preparation of antioxidant and anti-aging pharmaceuticals or cosmetics.

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