Soybean active peptide with hypoglycemic activity, and preparation method and application thereof

Abstract

The present application relates to soybean active peptides with hypoglycemic activity and a preparation method and application thereof, and belongs to the technical field of active peptide proteins. The active peptides are selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; the amino acid sequences of the SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 are respectively: GPFGT, DSRPLY, SGFGKL, IFGM. The soybean active peptides of the present application are verified by experiments to have hypoglycemic activity, are safe, have no toxic side effects, can be used as functional ingredients in hypoglycemic drugs, and have good potential and application prospects.

Description

Technical Field

[0001] This invention relates to soybean bioactive peptides with hypoglycemic activity, their preparation methods and applications, and belongs to the field of bioactive peptide protein technology. Background Technology

[0002] Diabetes mellitus is a common metabolic disease characterized by elevated blood glucose levels due to insufficient insulin secretion or inadequate glucose production. It is classified into type 1 and type 2. Type 1 diabetes, also known as insulin-dependent diabetes mellitus, is an autoimmune disease that causes a decrease or cessation of insulin secretion from the pancreatic beta cells. Type 2 diabetes, on the other hand, leads to inadequate glucose utilization by the body, resulting in an imbalance between insulin secretion and glucose absorption. Type 2 diabetes is often accompanied by symptoms associated with hyperglycemia, such as obesity, dyslipidemia, and hypertension. Sustained hyperglycemia or insulin resistance can disrupt the cellular redox system and lead to the production of reactive oxygen species, malondialdehyde, and ketone compounds. These substances attack fatty acids in biological membranes, causing lipid peroxidation and exacerbating cellular oxidative stress. These effects can lead to metabolic dysfunction and even death.

[0003] Most of the blood sugar-lowering drugs currently on the market are chemical drugs with significant side effects. Developing new formulations with blood sugar-lowering effects has broad application prospects.

[0004] Hypoglycemic peptides are polypeptides with hypoglycemic activity. They can be used in functional foods, pharmaceuticals, etc., and have broad application prospects.

[0005] Soybeans are rich in nutrients, with protein accounting for approximately 40%, and soy protein is a high-quality protein, rich in 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. Soy protein is a high-quality food protein for preparing hypoglycemic peptides. Soy protein can be degraded through enzymatic hydrolysis or microbial fermentation to produce bioactive soy peptides. Microbial fermentation for preparing bioactive soy peptides utilizes specific microorganisms and their produced proteases to hydrolyze soy protein. This method has 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 soy peptides obtained from soybean fermentation by different microorganisms vary.

[0006] *C. elegans*, with its short generation cycle, high reproductive rate, and ease of gene modification, is a preferred model organism for longevity, development, reproduction, and large-scale drug screening studies. *C. elegans* shares 60%–80% genetic homology with humans and shares conserved pathways in lipid accumulation and energy regulation. Lipids in *C. elegans* are easily stained, and triglyceride levels are readily measurable. In humans, a high-sugar diet generally leads to excessive lipid accumulation, ultimately resulting in obesity, dyslipidemia, hypertension, and other diabetic symptoms. Similarly, *C. elegans* induced by a high-sugar environment exhibits shortened lifespan, lipid accumulation, and lipid peroxidation. Therefore, *C. elegans* induced by a high-sugar environment is an ideal model organism for detecting hypoglycemic agents.

[0007] To date, various bioactive peptides have been prepared 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 hypoglycemic peptide sequences include VY, SFLLR, LLR, FH, RQLPR, KGF, and NFQ. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides soybean bioactive peptides with hypoglycemic activity, their preparation method, and applications. The soybean bioactive peptides obtained by this invention have the advantages of being safe and having no toxic side effects. The technical solution of this invention is as follows:

[0009] Soybean bioactive peptides with hypoglycemic activity, wherein the bioactive peptides are selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4 are respectively: Gly-Pro-Phe-Gly-Thr (GPFGT), Asp-Ser-Arg-Pro-Leu-Tyr (DSRPLY), Ser-Gly-Phe-Gly-Lys-Leu (SGFGKL), Ile-Phe-Gly-Met (IFGM).

[0010] The preparation method of the above-mentioned soybean bioactive peptides includes the following steps:

[0011] (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;

[0012] (2) Preparation of soybean active peptides by probiotic liquid fermentation of soybean protein: Bacillus coagulans was inoculated into the sterilized soybean protein solution obtained in step (1), 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.

[0013] Preferably, in step (2), the inoculation amount of Bacillus coagulans and Lactobacillus rhamnosus is 2% of the mass of the sterilized soybean protein solution.

[0014] (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 SPHP4.

[0015] (4) SPHP4 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;

[0016] 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.

[0017] Table 1 Elution conditions for ultra-high performance liquid chromatography

[0018]

[0019] 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.

[0020] Table 2 Tandem Mass Spectrometry Analysis Conditions

[0021]

[0022] (5) Bioinformatics prediction of each soybean peptide sequence:

[0023] 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.

[0024] 2) Peptide toxicity prediction analysis: ToxinPrep, based on the SVM algorithm, was used to perform potential toxicity prediction analysis on soybean peptides;

[0025] 3) Prediction of hypoglycemic activity of soybean peptides: For the soybean peptide sequences with PeptideRanker > 0.5 and no toxicity, AntiT2DMP-Pred was used to predict their hypoglycemic activity, and peptide sequences with predicted values ​​> 0.5 were screened; soybean hypoglycemic peptides GPFGT, DSRPLY, SGFGKL, and IFGM were obtained.

[0026] The present invention also includes the application of the above-mentioned soybean bioactive peptides in the preparation of hypoglycemic drugs.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] The inventors have for the first time screened four novel peptides from soybeans: GPFGT, DSRPLY, SGFGKL, and IFGM. Bioinformatics prediction results indicate that GPFGT, DSRPLY, SGFGKL, and IFGM have no toxic side effects and high biological activity. In vitro hypoglycemic experiments show that GPFGT, DSRPLY, SGFGKL, and IFGM have an IC50 inhibition rate of α-glucosidase. 50 The values ​​were 0.28±0.11 mg / mL, 0.24±0.05 mg / mL, 0.21±0.08 mg / mL, and 0.30±0.05 mg / mL, respectively. The in vivo hypoglycemic activity of soybean peptides was evaluated in the model organism *Caenorhabditis elegans*. In vivo hypoglycemic experiments showed that 1 mg / mL solutions of GPFGT, DSRPLY, SGFGKL, and IFGM prolonged the average lifespan of high-glycemic-induced *C. elegans* by 11.62%, 16.72%, 17.06%, and 14.00%, respectively, compared to the high-glycemic control group. Treatment of high-glycemic-induced nematodes with the hypoglycemic peptides GPFGT, DSRPLY, SGFGKL, and IFGM significantly reduced triglyceride and lipid content in the nematodes. In conclusion, both in vivo and in vitro experimental results confirm that the novel peptides GPFGT, DSRPLY, SGFGKL, and IFGM possess hypoglycemic activity. Therefore, novel soybean hypoglycemic peptides GPFGT, DSRPLY, SGFGKL, and IFGM can be used as functional ingredients in hypoglycemic drugs and have good application prospects. Attached Figure Description

[0029] Figure 1 The results of the α-glucosidase inhibition rate test of SPHP4 obtained in Example 1;

[0030] Figure 2 Survival curves of high-sugar nematode models treated with different concentrations of SPHP4. Detailed Implementation

[0031] 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.

[0032] The *Bacillus coagulans* and *Lactobacillus rhamnosus* used in the embodiments of this invention were both purchased from microbial culture collection centers. *Bacillus coagulans*, with accession number CCTCC AB 2018147, was purchased from the China Center for Type Culture Collection; *Lactobacillus rhamnosus*, with accession number CICC21006, was purchased from the China Industrial Microbial Culture Collection Center.

[0033] Example 1

[0034] Soybean bioactive peptides with hypoglycemic activity, wherein the bioactive peptides are selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4.

[0035] The preparation method of the above-mentioned soybean bioactive peptides includes the following steps:

[0036] (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.

[0037] (2) Prepare soybean active peptides by inoculating probiotics into the sterilized soybean protein solution: first inoculate with Bacillus coagulans (inoculation amount of 2%, w / w), and culture at 47℃ with shaking for 42 h, with a shaking speed of 150 rpm; then inoculate with Lactobacillus rhamnosus (inoculation amount of 2%, w / w), and culture at 37℃ with shaking for 36 h, with a shaking speed of 150 rpm;

[0038] (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 SPHP4.

[0039] (4) Mass spectrometry identification of SPHP4:

[0040] 1) The prepared SPHP4 was freeze-dried under vacuum and reconstituted with deionized water. Particles were removed by filtering the SPHP4 through 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.

[0041] 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.

[0042] 2) Mass spectrometry data analysis

[0043] 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).

[0044] 3) UPLC-ESI-TOF-MS / MS analysis revealed that the component peaks of soybean peptides were mainly concentrated at elution times of 0-5 and 45-60 min. Mass spectrometry analysis identified 137 peptide sequences with molecular weights ranging from 200-1000 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 hypoglycemic activity assays.

[0045] (5) Predictive analysis of the biological activity of soybean peptides:

[0046] 1) Prediction of the potential biological activity of peptides

[0047] 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 probability. 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. Bioinformatics prediction analysis results showed that 21 peptide sequences with PeptideRanker values ​​> 0.5 were obtained. Based on subsequent toxicity and hypoglycemic activity analyses, the peptide sequences GPFGT, DSRPLY, SGFGKL, and IFGM were selected for chemical synthesis and in vitro / in vivo validation, with PeptideRanker values ​​of 0.82, 0.52, 0.53, and 0.85, respectively (see Table 3).

[0048] Table 3. Mass spectrometry analysis and biological activity prediction results of soybean peptides

[0049]

[0050] 2) Prediction of the potential toxicity of soybean peptides

[0051] 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.

[0052] According to toxicity prediction analysis, all 21 peptide sequences with a PeptideRanker value > 0.5 were non-toxic bioactive peptides.

[0053] 3) Prediction of the antioxidant properties of soybean peptides

[0054] Soybean peptide sequences with a PeptideRanker > 0.5 and no toxicity were used to predict their hypoglycemic activity using AntiT2DMP-Pred (https: / / balalab-skku.org / AntiT2DMP-Pred / ). Fourteen peptide sequences with a prediction value > 0.5 were screened, and the peptide sequence with the highest prediction value was selected for subsequent experiments. The analysis results are shown in Table 4.

[0055] Table 4. Predictive analysis results of the hypoglycemic activity of soybean peptides

[0056]

[0057] 4) Bioinformatics analysis identified four soybean peptides with the following amino acid sequences: GPFGT, DSRPLY, SGFGKL, and IFGM, corresponding to SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, 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 is >95%.

[0058] Experimental Example 1

[0059] Assay of the in vitro hypoglycemic activity of SPHP4:

[0060] The hypoglycemic activity of SPHP4 was determined using an α-glucosidase inhibition rate assay. Unless otherwise specified, all pharmaceuticals and instruments used in this embodiment are commercially available.

[0061] Take SPHP4 and dilute it to 1 mg / mL. Mix 50 µl of 1 U / mL α-glucosidase with 100 µl of SPHP4 sample. Incubate at 37℃ for 10 min, then add 50 µl of 5 mM p-nitrophenyl-α-D-glucopyranoside as substrate. React at 37℃ for 30 min, then stop the reaction by adding 1 mL of 0.1 mol / L Na2CO3. A control group without sample solution and a blank group without substrate solution are also included. The α-glucosidase inhibition rate is calculated as: α-glucosidase inhibition rate (%) = [1 - (Ac-As) / (Ac-Ab)] × 100%

[0062] Where: Ac: absorbance of the control group (water + enzyme + substrate); As: absorbance of the sample group (sample + enzyme + substrate); Ab: absorbance of the blank group (water + substrate).

[0063] Calculations showed that the IC50 inhibition rate of SPHP4 against α-glucosidase was [value missing]. 50 The value was 0.3184 ± 0.025 mg / mL (e.g. Figure 1 (As shown).

[0064] Experimental Example 2

[0065] Animal studies evaluating the in vivo glycemic effect of SPHP4:

[0066] Synchronized L4-stage adult *C. elegans* were randomly selected and inoculated into high-glucose NGM petri dishes containing OP50 bacterial culture and 40 mM glucose, with 30 worms per group. Experimental and control groups were established. The experimental groups received 30.0 μL of SPHP4 solution (sterilized with a 0.22 μm filter) at concentrations of 0.30 mg / mL (low-dose), 0.60 mg / mL (medium-dose), and 1.20 mg / mL (high-dose), respectively. 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 high-glucose NGM petri dishes containing OP50 bacterial culture. The number of surviving and dead *C. elegans* worms was observed and recorded daily, with mortality determined by the worms' response to external stimuli. The experiment continued until all *C. elegans* worms had died. The average lifespan of the nematodes was calculated.

[0067] Survival curves of the high-sugar nematode model treated with different concentrations of SPHP4 are attached. Figure 2 The average lifespan of nematodes in the blank control group was 18.29±0.08 days, while that in the high-glucose model control group was 14.71±1.04 days. The average lifespan of *C. elegans* in the blank control group was significantly reduced (p<0.05), indicating successful model establishment. The lifespans of nematodes treated with low, medium, and high concentrations of SPHP4 were 15.41±1.14 days, 16.31±0.83 days, and 17.47±0.85 days, respectively. SPHP4 prolonged the average lifespan of high-glucose-induced nematodes by 4.76±0.68%, 10.88±1.50%, and 18.76±1.29%, respectively. The average lifespan of nematodes in the medium and high-dose groups was significantly increased (p<0.05), demonstrating that SPHP4 can restore the lifespan of *C. elegans* induced by high-glucose, and the restoration effect increases with increasing concentration.

[0068] Experimental Example 3

[0069] The in vitro hypoglycemic activity of the peptide was determined by the α-glucosidase inhibition rate assay.

[0070] The method and procedure for the α-glucosidase inhibition rate test are the same as those for Test Example 1.

[0071] Calculations showed that the IC50 inhibition rates of α-glucosidase from GPFGT, DSRPLY, SGFGKL, and IFGM were [missing data]. 50 The values ​​were 0.28±0.11 mg / mL, 0.24±0.05 mg / mL, 0.21±0.08 mg / mL, and 0.30±0.05 mg / mL, respectively. In vitro experiments confirmed that the novel soybean bioactive peptides GPFGT, DSRPLY, SGFGKL, and IFGM have hypoglycemic activity.

[0072] Test Example 4

[0073] (1) Effect of soybean peptides on the lifespan of high-sugar-induced nematodes:

[0074] Synchronized L4-stage adult *C. elegans* were randomly selected and inoculated into high-glucose NGM petri dishes containing OP50 bacterial culture and 40 mM glucose, with 30 worms per group. There were experimental groups, a high-glucose model control group, and a normal control group. The experimental group received 30.0 μL of a 1 mg / mL peptide solution sterilized via a 0.22 μm filter, while the control group received M9 buffer instead of the sample. Incubation was carried out 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 high-glucose NGM petri dishes containing OP50 bacterial culture. The number of surviving and dead *C. elegans* worms was observed and recorded daily, with mortality determined by the worms' response to external stimuli. The experiment continued until all *C. elegans* worms had died. The average lifespan of the nematodes was calculated.

[0075] The experimental results showed that the average lifespan of nematodes in the blank control group was 18.29±0.08 days, while that in the high-glucose model control group was 14.71±1.04 days. The average lifespan of *C. elegans* in the blank control group was significantly reduced (p<0.05), indicating successful model establishment. The average lifespan of *C. elegans* induced by high-glucose treatment with GPFGT, DSRPLY, SGFGKL, and IFGM was 16.42±0.63 days, 17.17±0.25 days, 17.22±0.18 days, and 16.77±0.33 days, respectively, which were 11.62%, 16.72%, 17.06%, and 14.00% longer than that in the high-glucose model control group, respectively. Biostatistical analysis showed that all differences were statistically significant. p <0.05). In vivo hypoglycemic assay results showed that all four peptides could significantly prolong the lifespan of high glucose-induced Caenorhabditis elegans, indicating that the four peptides have an in vivo hypoglycemic effect.

[0076] (2) Effect of soybean hypoglycemic peptides on triglyceride content in high-glucose-induced nematodes

[0077] The culture method was the same as (1). After 3 days of nematode culture, the nematodes were rinsed, homogenized, and then the triglyceride content was determined according to the instructions of the Nanjing Jiancheng Biotechnology Co., Ltd. triglyceride kit.

[0078] Triglycerides are an important component of fat and are widely distributed in intestinal and subcutaneous tissue cells. A high-sugar diet induces an increase in triglyceride levels in *C. elegans*. Experimental results showed that the triglyceride levels in the high-sugar control group and the normal control group were 0.64±0.08 and 0.27±0.02, respectively. Compared with the normal control group, high-sugar induced a significant increase in triglyceride levels in *C. elegans*. p<0.05. Treatment with glucose-lowering peptides GPFGT, DSRPLY, SGFGKL, and IFGM resulted in triglyceride levels in nematodes induced by high glucose levels of 0.44±0.03, 0.33±0.01, 0.31±0.02, and 0.41±0.08, respectively, all significantly lower than those in the high glucose-induced group. p <0.05).

[0079] (3) Effects of soybean hypoglycemic peptides on fat content in high-sugar-induced nematodes

[0080] Collect nematodes, wash with M9 buffer, centrifuge at 3000 rpm for 30 s, repeat until the nematodes are clean, discard the supernatant, add 150 μL of 1% paraformaldehyde, fix for 15 min; place the fixed nematodes in a -80℃ freezer for 2 min, then in a room temperature water bath for 1 min, repeat this freeze-thaw cycle 3 times, then wash 3 times with M9 buffer, discard the supernatant; add 1 mL of 60% isopropanol for dehydration for 10 min, centrifuge at 3000 rpm for 30 s, discard the supernatant; add 200 μL of Oil Red O working solution to each tube, stain in the dark for 30-60 min; wash 3 times with M9 buffer containing 0.01% Triton-X100, centrifuge, discard the supernatant, then transfer the nematodes to a 2% agarose gel, cover with a coverslip, photograph, and use ImageJ software to measure and calculate the relative staining area of ​​the nematodes. The calculation formula is: Relative staining area = (Staining area / Total nematode area) × 100%.

[0081] Lipid droplets are stored in the intestines and subcutaneous cells of *C. elegans*, and their outlines are clearly visible after staining with Oil Red O. A high-sugar diet induces an increase in fat content within *C. elegans*, and fat accumulation can be observed using Oil Red O staining.

[0082] The experimental results showed that the fat content of nematodes in the high-sugar control group and the normal control group was 72.82±5.93 and 8.72±3.09, respectively. Compared with the normal control group, the fat content of *C. elegans* was significantly increased under high-sugar inducement. p <0.05, the body fat content of nematodes treated with glucose-lowering peptides GPFGT, DSRPLY, SGFGKL, and IFGM were 27.03±4.16, 20.46±3.08, 20.15±3.12, and 23.72±4.37, respectively, which were significantly lower than those in the high glucose-induced group. p <0.05).

[0083] The above in vivo and in vitro analysis results indicate that soybean hypoglycemic peptides GPFGT, DSRPLY, SGFGKL, and IFGM have hypoglycemic activity and can be used to develop hypoglycemic related drugs.

[0084] 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 hypoglycemic activity, wherein the bioactive 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 respectively: Gly-Pro-Phe-Gly-Thr (GPFGT), Asp-Ser-Arg-Pro-Leu-Tyr (DSRPLY), and Ser-Gly-Phe-Gly-Lys-Leu (SGFGKL).

2. A method for preparing soybean bioactive peptides with hypoglycemic activity, 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 coagulans was inoculated into the sterilized soybean protein solution obtained in step (1), 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 coagulans was preserved with the accession number: CCTCC AB 2018147; the Lactobacillus rhamnosus was preserved with the accession number: CICC21006; the inoculation amount of Bacillus coagulans and Lactobacillus rhamnosus was 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 SPHP4. (4) SPHP4 was filtered to remove particles, and the soybean peptide sequence was obtained by mass spectrometry identification using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-TOF-MS / MS). (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 soybean peptides; 3) Prediction of hypoglycemic activity of soybean peptides: For the soybean peptide sequences with PeptideRanker > 0.5 and no toxicity, AntiT2DMP-Pred was used to predict their hypoglycemic activity, and peptide sequences with predicted values ​​> 0.5 were screened; soybean active peptides GPFGT, DSRPLY, SGFGKL and IFGM were obtained.

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), SPHP4 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 UPLC 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 hypoglycemic drugs.

9. The use of soybean bioactive peptides obtained by the preparation method according to any one of claims 2-7 in the preparation of hypoglycemic drugs.

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