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

Abstract

The invention relates to a soybean active peptide with hypoglycemic 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, SEQ ID NO. 3 or SEQ ID NO. 4; the amino acid sequences of the SEQ ID NO.1, the SEQ ID NO.2, the SEQ ID NO.3 and the SEQ ID NO.4 are respectively GPFGT, DSRPLY, SGFGKL and IFGM (Induced Factor Growth Factor). Experiments prove that the soybean active peptide has hypoglycemic activity, is safe and free of toxic and side effects, can be used as a functional component in hypoglycemic drugs, and has good potential and application prospects.

Description

Technical Field

[0001] The present invention relates to soybean active peptide with hypoglycemic activity and a preparation method and application thereof, belonging to the technical field of active peptide proteins. Background Art

[0002] Diabetes is a common metabolic disease characterized by elevated blood sugar levels due to insufficient insulin secretion or insufficient blood sugar. It is divided into type I and type II. Type I diabetes is insulin-dependent diabetes, an autoimmune disease that causes a decrease or cessation of insulin secretion by the pancreatic beta cells; type II diabetes results in insufficient glucose utilization by the body, resulting in an imbalance between insulin secretion and blood sugar absorption. Type II diabetes is also often manifested by symptoms such as excessive obesity, dyslipidemia, and hypertension associated with hyperglycemia. Persistent hyperglycemia or insulin resistance can lead to damage to the cell's redox system and the production of reactive oxygen species, malondialdehyde, and ketone compounds. These substances attack fatty acids in biological membranes, causing lipid peroxidation and exacerbating the cell's oxidative stress state. These effects can lead to metabolic dysfunction and even death.

[0003] Currently, most of the blood sugar lowering drugs sold on the market are chemical drugs with serious side effects. The development of new preparations with blood sugar lowering effects has broad application prospects.

[0004] Hypoglycemic peptides refer to polypeptides with hypoglycemic activity, which can be used as functional foods, medicines, etc. and have broad application prospects.

[0005] Soybeans are highly nutritious, with protein comprising approximately 40% of the protein content. 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 is a high-quality food protein for the production of glucose-lowering peptides. Soy protein can be degraded by enzymatic hydrolysis or microbial fermentation to produce bioactive soy peptides. Microbial fermentation for the production of bioactive soy peptides utilizes specific microorganisms and the proteases they produce to hydrolyze soy protein. This method 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 qualities, 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 vary in sequence and activity.

[0006] Caenorhabditis elegans, with its short generation cycle, high reproductive rate, and ease of genetic modification, is a preferred model organism for studies of longevity, development, reproduction, and large-scale drug screening. C. elegans shares 60% to 80% genetic homology with humans and shares conserved pathways for fat accumulation and energy regulation. C. elegans' lipids are easily stained, and triglyceride levels in the worms are also easily measured. In humans, a high-sugar diet generally leads to excessive lipid accumulation, ultimately causing symptoms of diabetes such as obesity, dyslipidemia, and hypertension. Similarly, C. elegans induced in a high-sugar environment exhibits shortened lifespan, lipid accumulation, and lipid peroxidation. Therefore, C. elegans induced in a high-sugar environment is an ideal model organism for testing hypoglycemic substances.

[0007] Soybean active peptides have been prepared from soy protein degradation products, including soy hypoglycemic peptides, soy immune peptides, soy anti-inflammatory peptides, soy blood pressure lowering peptides, soy lipid lowering peptides, soy anticancer peptides, and soy antioxidant peptides. Reported soy hypoglycemic peptide sequences include VY, SFLLR, LLR, FH, RQLPR, KGF, and NFQ. Summary of the Invention

[0008] In response to the problems of the above-mentioned prior art, the present invention provides a soybean active peptide with hypoglycemic activity, a preparation method thereof, and its application. The soybean active peptide obtained by the present invention has the advantages of being safe and having no toxic side effects. The technical solution of the present invention is as follows: A soybean active peptide with hypoglycemic activity, wherein the active peptide is 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: 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), respectively.

[0009] 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 coagulans into the sterilized soybean protein solution obtained in step (1), culture at 47°C with shaking for 42 h, and the shaker speed is 100-200 rpm; then inoculate Lactobacillus rhamnosus, culture at 37°C with shaking for 36 h, and the shaker speed is 100-200 rpm; obtain fermentation liquid; Preferably, in step (2), the inoculation amount of Bacillus coagulans and Lactobacillus rhamnosus is 2% of the mass of the sterilized soy protein solution.

[0010] (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 SPHP4. (4) SPHP4 was filtered through a 0.22 μm aqueous syringe filter to remove particles, and mass spectrometry identification was performed 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.

[0011] Table 1 Ultra-high performance liquid chromatography elution conditions

[0012] 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.

[0013] Table 2 Tandem mass spectrometry analysis conditions

[0014] (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 was used to predict the potential toxicity of soybean peptides based on the SVM algorithm; 3) Prediction of hypoglycemic activity of soybean peptides: For the aforementioned soybean peptide sequences with PeptideRanker > 0.5 and no toxicity, AntiT2DMP-Pred was used to predict their hypoglycemic activity, and peptide sequences with prediction values ​​> 0.5 were screened; the soybean hypoglycemic peptides GPFGT, DSRPLY, SGFGKL, and IFGM were obtained.

[0015] The present invention also includes the use of the soybean active peptide in the preparation of hypoglycemic drugs.

[0016] Compared with the prior art, the present invention has the following advantages: The inventors screened and obtained four new peptides, GPFGT, DSRPLY, SGFGKL, and IFGM, from soybeans for the first time. Bioinformatics prediction results showed that GPFGT, DSRPLY, SGFGKL, and IFGM had no toxic side effects and high biological activity. In vitro hypoglycemic experiments showed that the α-glucosidase inhibition rate IC of GPFGT, DSRPLY, SGFGKL, and IFGM was 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 soybean peptides were evaluated for their in vivo hypoglycemic activity in the model organism Caenorhabditis elegans. In vivo hypoglycemic assays showed that 1 mg / mL solutions of GPFGT, DSRPLY, SGFGKL, and IFGM extended the average lifespan of C. elegans induced by high glucose by 11.62%, 16.72%, 17.06%, and 14.00%, respectively, compared to the high glucose control group. Treatment of high glucose-induced nematodes with the hypoglycemic peptides GPFGT, DSRPLY, SGFGKL, and IFGM significantly reduced triglyceride and lipid content. In summary, both in vitro and in vivo results confirm the hypoglycemic activity of the novel peptides GPFGT, DSRPLY, SGFGKL, and IFGM. Therefore, the new soybean glucose-lowering peptides GPFGT, DSRPLY, SGFGKL, and IFGM can be used as functional ingredients in glucose-lowering drugs and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The α-glucosidase inhibition test results of SPHP4 obtained in Example 1; Figure 2 Survival curves of the high-sugar nematode model treated with different concentrations of SPHP4. DETAILED DESCRIPTION

[0018] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements fall within the scope of protection of the present invention.

[0019] The Bacillus coagulans and Lactobacillus rhamnosus used in the examples of the present invention were both purchased from bacterial culture collection centers. Bacillus coagulans, classified as Bacillus coagulans, with a collection number of CCTCC AB 2018147, was purchased from the China Center for Type Culture Collection; Lactobacillus rhamnosus, classified as Lactobacillus rhamnosus, with a collection number of CICC21006, was purchased from the China Center for Industrial Microbiological Culture Collection.

[0020] Example 1 A soybean active peptide with hypoglycemic activity, wherein the active peptide is selected from SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0021] 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 coagulans was inoculated (inoculation amount was 2%, w / w), and the culture was shaken at 47°C for 42 h, with a shaking speed of 150 rpm; then, Lactobacillus rhamnosus was inoculated (inoculation amount was 2%, w / w), and the culture was shaken at 37°C for 36 h, with a shaking speed of 150 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 SPHP4. (4) Mass spectrometry identification of SPHP4: 1) The prepared SPHP4 was freeze-dried in a vacuum oven and reconstituted in deionized water. The SPHP4 was filtered through a 0.22 μm aqueous syringe filter to remove particulates. The product was then identified by mass spectrometry using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-TOF-MS / MS). The UPLC mobile phase A consisted of an aqueous solution containing 0.1% formic acid, and the mobile phase B consisted of an acetonitrile solution containing 0.1% formic acid. The elution time was 60 minutes, 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.

[0022] 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.

[0023] 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).

[0024] 3) UPLC-ESI-TOF-MS / MS analysis revealed that the soy peptide component peaks were primarily concentrated at elution times 0-5 and 45-60 min. Mass spectrometry analysis identified 137 peptide sequences with molecular weights ranging from 200 to 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 hypoglycemic activity.

[0025] (5) Prediction and analysis of biological activity of soybean peptides: 1) Prediction of potential biological activity of peptides The potential biological activities of the obtained peptide sequences were analyzed using the PeptideRanker online platform (http: / / distilldeep.ucd). Peptides were ranked according to 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. Bioinformatics prediction analysis revealed 21 peptide sequences with PeptideRanker values ​​greater than 0.5. Combined with subsequent toxicity and hypoglycemic activity analyses, the peptide sequences GPFGT, DSRPLY, SGFGKL, and IFGM were selected for chemical synthesis and in vitro and in vivo validation. Their PeptideRanker values ​​were 0.82, 0.52, 0.53, and 0.85, respectively (see Table 3).

[0026] Table 3 Mass spectrometry analysis and biological activity prediction analysis results of soybean peptides

[0027] 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.

[0028] After toxicity prediction analysis, 21 peptide sequences with PeptideRanker values ​​greater than 0.5 were all non-toxic active peptides.

[0029] 3) Prediction of antioxidant activity of soybean peptides The identified soybean peptide sequences with a PeptideRanker score greater than 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 prediction values ​​greater than 0.5 were screened, and the peptide sequence with the highest prediction value was selected for subsequent testing. The analysis results are shown in Table 4.

[0030] Table 4 Prediction and analysis results of hypoglycemic activity of soybean peptides

[0031] 4) Bioinformatics analysis identified four soybean peptides with amino acid sequences: GPFGT, DSRPLY, SGFGKL, and IFGM (SEQ ID NOs. 1, 2, 3, and 4). 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%.

[0032] Test Example 1 In vitro hypoglycemic activity assay of SPHP4: The hypoglycemic activity of SPHP4 was determined using an α-glucosidase inhibition test. Unless otherwise specified, the drugs and instruments used in this example can be obtained commercially.

[0033] SPHP4 was diluted to 1 mg / mL. 50 µl of 1 U / mL α-glucosidase was mixed with 100 µl of SPHP4 sample and incubated at 37°C for 10 minutes. Then, 50 µl of 5 mM p-nitrophenyl-α-D-glucopyranoside was added as the substrate. The reaction was incubated at 37°C for 30 minutes, and then terminated by adding 1 mL of 0.1 mol / L Na₂CO₃. A control group containing no sample and a blank group containing no substrate were also prepared. The α-glucosidase inhibition rate was calculated as follows: α-glucosidase inhibition rate (%) = [1-(Ac-As) / (Ac-Ab)] × 100% 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).

[0034] The α-glucosidase inhibition rate of SPHP4 was calculated to be IC 50 The value is 0.3184±0.025 mg / mL (e.g. Figure 1 shown).

[0035] Test Example 2 Animal experiments evaluated the hypoglycemic effect of SPHP4 in vivo: Synchronized L4-stage Caenorhabditis elegans adults were randomly selected and plated on high-glucose NGM plates containing OP50 bacterial lawns and 40 mM glucose, with 30 worms per group. Experimental and control groups were established. The experimental groups were sterilized with a 0.22 μm filter and added 30.0 μL of SPHP4 solution at concentrations of 0.30 mg / mL (low-dose group), 0.60 mg / mL (medium-dose group), or 1.20 mg / mL (high-dose group). The 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 high-glucose NGM plates containing OP50 bacterial lawns. The number of worms alive and dead was observed and recorded daily. The worms' response to external stimuli was used as the criterion for death. The experiment was terminated when all worms died. The average lifespan of the worms was calculated.

[0036] The survival curves of the high-sugar nematode model treated with different concentrations of SPHP4 are shown in the attached figure. Figure 2 The average lifespan of C. elegans in the blank control group was 18.29±0.08 d, while that in the high-glucose model control group was 14.71±1.04 d. The average lifespan of C. elegans in the blank control group was significantly reduced (p<0.05), indicating successful model establishment. The lifespans of C. elegans treated with low, medium, and high concentrations of SPHP4 were 15.41±1.14 d, 16.31±0.83 d, and 17.47±0.85 d, respectively. SPHP4 extended the average lifespan of C. elegans induced by high glucose by 4.76±0.68%, 10.88±1.50%, and 18.76±1.29%, respectively. The average lifespan of C. elegans 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.

[0037] Test Example 3 Determination of in vitro hypoglycemic activity of peptides by α-glucosidase inhibition test The α-glucosidase inhibition test method and steps are the same as those in Test Example 1.

[0038] After calculation, the α-glucosidase inhibition rate IC of GPFGT, DSRPLY, SGFGKL and IFGM 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 active peptides GPFGT, DSRPLY, SGFGKL, and IFGM have hypoglycemic activity.

[0039] Test Example 4 (1) Effect of soybean peptide on the lifespan of nematodes induced by high sugar: Synchronized L4-stage Caenorhabditis elegans adults were randomly selected and inoculated onto high-glucose NGM plates containing OP50 bacterial lawns and 40 mM glucose, with 30 worms per group. Experimental, high-glucose model, and normal controls were established. The experimental groups were treated with 30.0 μL of a 1 mg / mL peptide solution sterilized with a 0.22 μm filter, while the control groups were treated with M9 buffer instead of sample. The cells were incubated at 20°C. The day of transfer was designated as day 0. At the same time each day, C. elegans were transferred to a new high-glucose NGM plate containing OP50 bacterial lawns. The number of worms that survived and died was recorded daily. The experiment was terminated when all worms died, based on their response to external stimuli. The mean lifespan of the worms was calculated.

[0040] The experimental results showed that the average lifespan of C. elegans in the blank control group was 18.29±0.08 days, while the average lifespan of C. elegans 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 that the model was successfully established. The average lifespan of C. elegans induced by high glucose treated 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 those in the high-glucose model control group, respectively. Biostatistical analysis showed that there were significant differences ( p <0.05). The results of the in vivo hypoglycemic test showed that all four peptides could significantly prolong the lifespan of C. elegans induced by high glucose, indicating that the four peptides have hypoglycemic effects in vivo.

[0041] (2) Effect of soybean hypoglycemic peptide on triglyceride content in nematodes induced by high glucose The culture method was the same as (1). After 3 days of culture, the nematodes were rinsed and homogenized, and then the triglyceride content was determined according to the instructions of the triglyceride kit of Nanjing Jiancheng Biological Co., Ltd.

[0042] Triglycerides are an important component of fat and are widely present in the intestinal and subcutaneous tissue cells. A high-sugar diet can lead to an increase in triglyceride content in C. elegans. The experimental results showed that the triglyceride content of nematodes in the high-sugar control group and the normal control group was 0.64±0.08 and 0.27±0.02, respectively. Compared with the normal control group, high sugar-induced triglyceride content in C. elegans increased significantly ( p<0.05). The triglyceride levels in nematodes induced by high glucose were 0.44±0.03, 0.33±0.01, 0.31±0.02, and 0.41±0.08, respectively, which were significantly lower than those in the high glucose-induced group ( p <0.05).

[0043] (3) Effect of soybean hypoglycemic peptide on fat content in nematodes induced by high sugar Nematodes were collected, washed with M9 buffer, and centrifuged at 3000 rpm for 30 s. This process was repeated until the nematodes were completely washed. The supernatant was discarded, and 150 μL of 1% paraformaldehyde was added for fixation for 15 min. The fixed nematodes were placed in a -80°C freezer for 2 min, then placed in a room temperature water bath for 1 min. This freeze-thaw cycle was repeated three times, followed by three washes with M9 buffer, and the supernatant was discarded. The tubes were dehydrated with 1 mL of 60% isopropanol for 10 min, centrifuged at 3000 rpm for 30 s, and the supernatant discarded. 200 μL of Oil Red O working solution was added to each tube, and the tubes were stained in the dark for 30-60 min. The tubes were washed three times with M9 buffer containing 0.01% Triton-X100, centrifuged, and the supernatant discarded. The nematodes were then transferred to 2% agarose gel pads, covered with a coverslip, and photographed. The relative stained area of ​​the nematodes was measured and calculated using ImageJ software. The formula for calculation was: relative stained area = (stained area / total nematode area) × 100%.

[0044] Lipid droplets are stored in the intestinal 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 in C. elegans, and this fat accumulation can be observed using Oil Red O staining.

[0045] 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, high sugar induced a significant increase in the fat content of C. elegans ( p <0.05), the body fat contents of nematodes induced by high glucose 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).

[0046] The above in vivo and in vitro analysis results show that soybean glucose-lowering peptides GPFGT, DSRPLY, SGFGKL, and IFGM have glucose-lowering activity and can be used to develop glucose-lowering related drugs.

[0047] 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 hypoglycemic activity, wherein the active peptide is 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, and SEQ ID NO. 4 are: Gly-Pro-Phe-Gly-Thr (GPFGT), Asp-Ser-Arg-Pro-Leu-Tyr (DSRPLY), Ser-Gly-Phe-Gly-Lys-Leu (SGFGKL), and Ile-Phe-Gly-Met (IFGM), 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 coagulans into the sterilized soybean protein solution obtained in step (1), culture at 47°C with shaking for 42 h, and the shaker speed is 100-200 rpm; then inoculate Lactobacillus rhamnosus, culture at 37°C with shaking for 36 h, and the shaker speed is 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 SPHP4. (4) SPHP4 was filtered to remove particles, and mass spectrometry identification was performed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-TOF-MS / MS) 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 was used to predict the potential toxicity of soybean peptides based on the SVM algorithm; 3) Prediction of hypoglycemic activity of soybean peptides: For the aforementioned soybean peptide sequences with PeptideRanker > 0.5 and no toxicity, AntiT2DMP-Pred was used to predict their hypoglycemic activity, and peptide sequences with prediction values ​​> 0.5 were screened; the soybean active peptides GPFGT, DSRPLY, SGFGKL, and IFGM were obtained.

3. The preparation method according to claim 2, characterized in that The material-liquid ratio of soy protein to distilled water in step (1) is 1:20 (w / w).

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 step (2), the inoculation amount of 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), SPHP4 is filtered using a 0.22 μm water-based syringe filter.

7. The preparation method according to claim 2, wherein In step (4), 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 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 hypoglycemic drugs.

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 hypoglycemic drugs.

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