Yeast protein product with blood sugar reducing function and preparation method thereof

Abstract

The invention relates to a yeast protein product with a blood sugar reducing function and a preparation method thereof, the protein content of the yeast protein product is greater than 75%, the yeast protein product contains specific amino acid sequences FPSPM, HFPF and AWLPR, and the preparation method comprises the following steps: fermenting yeast; preparing a suspension from the fermented yeast thalli, performing heat preservation treatment under a certain condition, and collecting a precipitate; preparing the precipitate into a 2%-10% aqueous solution, and homogenizing and breaking walls for 2-3 times by adopting a high-pressure homogenizer under the pressure of 500-1500Bar; adjusting the pH value, adding 5-50 U / mL glucanase and 5-50 U / mL cellulase at the temperature of 30-60 DEG C, and carrying out enzymolysis to obtain an enzymatic hydrolysate; and carrying out solid-liquid separation on the enzymatic hydrolysate, washing, collecting the precipitate, and drying to obtain a yeast protein product. The yeast protein product prepared by the preparation method disclosed by the invention can promote secretion of GLP-1 and other intestinal insulinotropic hormones and inhibit the activity of DPP-4 so as to reduce hydrolysis of GLP-1 after being subjected to digestive tract enzymolysis, has the effects of reducing blood sugar and reducing body weight, and is free of toxic and side effects, simple in preparation method, mild in condition and suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a yeast protein product with blood sugar-lowering function and its preparation method. Background Technology

[0002] Diabetes mellitus is a metabolic disease caused by insufficient insulin secretion or insulin resistance, leading to abnormally high blood sugar levels. Long-term hyperglycemia can easily lead to various long-term health complications, including heart disease, stroke, and systemic circulatory disorders. This disease is also highly prevalent in China. A research report released on June 2, 2025, showed that the estimated number of diabetes patients in China reached 233 million in 2023, with an age-standardized prevalence of 13.7%. Currently, drug therapy is one of the core methods of diabetes management, mainly divided into two categories: oral hypoglycemic agents and injectable hypoglycemic agents. Injectable drugs include insulin and glucagon-like peptide-1 receptor agonists (GLP-1); oral hypoglycemic agents cover major categories such as biguanides, sulfonylureas, and sodium-glucose cotransporter 2 inhibitors.

[0003] Obesity has become one of the major health challenges facing China today.

[0004] Glucagon-like peptide-1 (GLP-1) is an incretin composed of 37 amino acids. Its core physiological functions include stimulating insulin secretion, protecting pancreatic β-cells, inhibiting glucagon secretion, delaying gastric emptying, and reducing food intake, ultimately achieving the dual effects of lowering blood sugar and reducing weight. However, GLP-1 secretion is impaired in diabetic and obese patients, with significantly lower postprandial GLP-1 secretion compared to normal individuals, resulting in weakened control of postprandial blood glucose. Liraglutide and semaglutide, as GLP-1 receptor agonists, have been approved for the treatment of type 2 diabetes and obesity.

[0005] However, natural GLP-1 is readily inactivated or degraded by enzymes such as dipeptidyl peptidase-4 (DPP-4), with a half-life of only about 1.5 minutes, severely limiting its glycemic regulatory efficacy. DPP-4 is a serine peptidase located on the cell surface, belonging to the membrane protein family, and widely distributed in tissues such as the kidneys, connective tissue, gastrointestinal tract, and lymph nodes. It can rapidly degrade incretin hormones such as GLP-1. Therefore, developing food components that promote GLP-1 secretion or inhibit DPP-4 activity has a good function of lowering blood sugar and improving weight. Long-acting GLP-1 derivatives and GLP-1 receptor agonists have become the focus of research in the field of diabetes control due to their superior secretin effect. These GLP-1 analogs and receptor agonists are widely recognized in existing technologies. GLP-1 belongs to the incretin hormone family, and its receptor signaling plays an important physiological role in maintaining glucose homeostasis.

[0006] In the prior art, patent CN119212574A discloses a hydrolyzed collagen preparation with hypoglycemic effect. This preparation is obtained by enzymatic hydrolysis of collagen-containing raw materials using a combination of proteases, and is suitable as a dietary supplement to help improve hyperglycemia and related risk factors. Patent JP2012116773 discloses a collagen peptide derived from fish scales and / or fish skin collagen, which is prepared by protease treatment and can increase the level of insulin-like growth factor 1 (IGF-1). CN 202210528272.3 discloses a DPP-4 inhibitory peptide derived from yak collagen, with the sequence MGPR, which has DPP-4 inhibitory activity.

[0007] Although existing technologies have developed various active incretin analogs, these products are mostly used as drugs, which has many limitations. Therefore, developing food-derived products with clearly defined activity and better safety that can act on incretins, and providing safer and more effective supplementary foods for obesity, diabetes and their complications, has become an important issue that urgently needs to be addressed in this field. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a yeast protein product with blood sugar lowering function and its preparation method. The yeast protein product contains a specific amino acid sequence, which can significantly promote the secretion of glucagon-like peptide-1 (GLP-1) in gastrointestinal digestive products and inhibit DPP-4 activity to reduce the breakdown of GLP-1, thereby having the effect of lowering blood sugar and improving weight.

[0009] This invention is achieved through the following technical solution: A yeast protein product with blood sugar lowering function has a protein content of more than 75% and contains specific amino acid sequences FPSPM (Phe-Pro-Ser-Pro-Met), HFPF (His-Phe-Pro-Phe), and AWLPR (Ala-Trp-Leu-Pro-Arg). FPSPM is shown in SEQ ID NO: 1, HFPF is shown in SEQ ID NO: 2, and AWLPR is shown in SEQ ID NO: 3.

[0010] A method for preparing the above-mentioned yeast protein product with blood sugar lowering function includes the following steps: S1. Yeast cell fermentation: After culturing yeast cells on slant culture medium, one loop of activated cells is inoculated into the planting medium and cultured on a shaker at 25-35℃ and 150-250 r / min for 12-24 h. Then, 5% of the inoculum is inoculated into the fermentation medium and cultured on a shaker at 25-35℃ and 150-250 r / min for 12-24 h. After centrifugation, washing, and centrifugation to collect the precipitate, the fermented yeast cells are obtained. S2. Heat treatment of yeast cells: Prepare a suspension of fermented yeast cells obtained in step S1 with a weight percentage of 5%-15%, adjust the pH to 7.0-9.0, set the temperature to 60-100℃, keep it at this temperature for 30-240 minutes, and then centrifuge to separate the precipitate. S3. High-pressure homogenization and cell disruption: Prepare an aqueous solution with a weight percentage of 2%-10% from the precipitate obtained in step S2, and homogenize and disrupt the cell walls 2-3 times using a high-pressure homogenizer at a pressure of 500-1500 Bar. S4. Enzymatic hydrolysis: Adjust the pH of the homogenized, cell-wall-broken solution obtained in step S3 to 5.0-7.0 and the temperature to 30-60℃. Add 5-50 U / mL of dextranase and 5-50 U / mL of cellulase and hydrolyze for 4-24 hours to obtain the enzymatic hydrolysate. S5. Solid-liquid separation: The enzymatic hydrolysate obtained in step S4 is subjected to solid-liquid separation, the precipitate is collected, and the precipitate is washed 2-3 times with the same volume of hot water at 40-60°C as the enzymatic hydrolysate. The precipitate is collected and dried to obtain the yeast protein product with the function of lowering blood sugar.

[0011] Furthermore, in step S1, the yeast strains used for yeast protein production include, but are not limited to, *Kluyveromyces*.

[0012] Further, in step S1, the slant culture medium includes YPD medium and agar, wherein the weight percentage of the agar is 2%, and the YPD medium includes the following components in weight percentage: yeast extract 1%, peptone 2%, and glucose 2%; The seed culture medium includes YPD medium, which comprises the following components in weight percentage: yeast extract 1%, peptone 2%, and glucose 2%; The fermentation medium comprises the following components by weight percentage: glucose 6%, yeast extract 0.5%-1.0%, (NH4)2SO4 0.4%, KH2PO4 0.6%, K2SO4 0.1%, and the pH of the fermentation medium is 5.5; The slant culture medium, seed culture medium and fermentation culture medium were all sterilized under saturated steam at 121℃ for 15 min.

[0013] Furthermore, in step S2, the weight percentage of the prepared suspension is 6%-8%, the pH of the suspension is adjusted to 7.5-8.5, the holding temperature is 80-90℃, and the holding time is 60-120 min.

[0014] Furthermore, in step S3, the weight percentage of the prepared aqueous solution is 5%-8%, and the pressure for cell disruption is 800-1200 Bar.

[0015] Furthermore, in step S4, the enzymatic hydrolysis temperature is 40-55℃ and the enzymatic hydrolysis time is 8-12h.

[0016] Furthermore, it also includes a step S6 for identifying the amino acid sequence, which includes: S6-1. The yeast protein product is digested with simulated gastric juice to obtain product G-KP, and G-KP is then digested with simulated intestinal juice to obtain product GI-KP. S6-2 and GI-KP peptide structures were identified by nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS). The raw data were processed and analyzed using PEAKS software. Then, after ALC (average local confidence level) scoring and DPP-4 inhibitory activity evaluation in the BIOPEP database, 42 peptides with good DPP-4 inhibitory activity were screened out. S6-3. The toxicity, solubility, intestinal absorption, and sensitization of the 42 peptides were predicted using the ToxinPred, SwissADME, and Allergen FP databases, respectively. Eight peptides with excellent properties were screened out, namely FPSPM (Phe-Pro-Ser-Pro-Met), HFPF (His-Phe-Pro-Phe), FKEWL (Phe-Lys-Glu-Trp-Leu), AWLPR (Ala-Trp-Leu-Pro-Arg), GPAGPQGPR (Gly-Pro-Ala-Gly-Pro-Gln-Gly-Pro-Arg), GWV (Gly-Trp-Val), WLGGH (Trp-Leu-Gly-Gly-His), and LPFP (Leu-Pro-Phe-Pro). S6-4. Using Autodock Vina software, the eight selected peptides were subjected to molecular docking simulation with DDP-4. Finally, three peptides with low binding energies to DPP-4 were selected: FPSPM, HFPF, and AWLPR.

[0017] Further, in step S6-2, the method for identifying the peptide structure of GI-KP using nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS) is as follows: GI-KP is dissolved in mobile phase A (0.1% trifluoroacetic acid aqueous solution) to prepare a 0.5 mg / mL solution. After desalting using a C18 column, the solution is filtered through a 0.22 µm microporous membrane and then introduced into nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS). The chromatographic column is a PepMap C18, mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, and mobile phase B is a 0.1% formic acid acetonitrile solution. The separation gradient is as follows: mobile phase B (0.1% formic acid acetonitrile solution) increases from 5% to 35% within 70 min. The sample loading volume is 3 μL, the chromatographic flow rate is 300 nL / min, and the column temperature is 60 °C. Mass spectrometry is performed using ion spray at a voltage of 2.3 kV. The method for selecting 42 peptides with good DPP-4 inhibitory activity after sequentially using ALC (Average Local Confidence) scoring and DPP-4 inhibitory activity evaluation in the BIOPEP database is as follows: The resolved peptides are scored using PeptideRanker with a score threshold of 0.8, from which 128 peptides with high potential are selected; then these 128 peptides are compared with the DPP-4 inhibitory activity in the BIOPEP database, and finally 42 peptides with good DPP-4 inhibitory activity are selected.

[0018] Further, in step S6-4, the method of using Autodock Vina software to simulate molecular docking between the eight screened peptides and DDP-4 is as follows: obtain the crystal structure of DPP-4 from the PDB database, and simultaneously use ChemBio3D software to draw the three-dimensional structure of each peptide to be screened; import the crystal structure of DPP-4 and the three-dimensional structure of the peptides into Autodock Vina software, and after dehydration and hydrogenation steps, start the molecular docking simulation, and screen active peptides according to the strength of binding sites and binding energies.

[0019] This invention uses *Kluyveromyces martensii* as the raw material for yeast protein products. Through fermentation, heat treatment (to remove nucleic acids and water-soluble polysaccharides), high-pressure homogenization, enzymatic hydrolysis (to remove yeast polysaccharides), separation, and drying, a yeast protein composition product is obtained. This product has a protein content of over 75%. The yeast protein composition product contains specific amino acid sequences: FPSPM (Phe-Pro-Ser-Pro-Met), HFPF (His-Phe-Pro-Phe), and AWLPR (Ala-Trp-Leu-Pro-Arg). Experimental results confirm its effectiveness. These peptides are found in the products of digestive enzyme hydrolysis, and can promote the secretion of incretin hormones such as GLP-1, inhibit DPP-4 activity to reduce GLP-1 hydrolysis, thus having the effects of lowering blood sugar and weight, and without toxic side effects. The preparation method of the yeast protein composition product is simple and mild, suitable for large-scale production. Peptides with good inhibitory effects on DPP-4 were screened using techniques such as liquid chromatography-tandem mass spectrometry, ALC scoring, DPP-4 inhibitory activity comparison, and molecular docking simulation, providing technical support for the detection of the final yeast protein composition product. Attached Figure Description

[0020] Figure 1 The survival rate of STC-1 cells at different concentrations of yeast protein products and their mimicry intestinal digestion products.

[0021] Figure 2 The secretion levels of GLP-1 and CCk in yeast protein products and their mimicry intestinal digestion products at different concentrations.

[0022] Figure 3 This is the mass spectrum of the FPSPM peptide.

[0023] Figure 4 This is the mass spectrum of the HFPF peptide.

[0024] Figure 5 This is the mass spectrum of the AWLPR peptide.

[0025] Figure 6 Global and detailed images of the docking of the FPSPM peptide with DPP-4.

[0026] Figure 7 Global and detailed images of the docking of the HFPF peptide with DPP-4.

[0027] Figure 8 Global and detailed images of the AWLPR peptide docking with DPP-4.

[0028] Figure 9 The half-maximal inhibitory concentrations (IC50) of FPSPM, HFPF, and AWLPR peptides for DPP-4 inhibition are given. 50 value). Detailed Implementation

[0029] Example 1: Yeast cell fermentation Culture medium: Slant culture medium: YPD medium (1% yeast extract, 2% peptone, 2% glucose), 2% agar.

[0030] Seed culture medium: YPD medium (1% yeast extract, 2% peptone, 2% glucose).

[0031] Fermentation medium: glucose 6%, yeast extract 0.5%, (NH4)2SO4 0.4%, KH2PO4 0.6%, K2SO4 0.1%, pH 5.5.

[0032] All culture media were sterilized at 121℃ under saturated steam for 15 minutes.

[0033] After culturing *Kluyveromyces martensii* (strain preservation number CGMCC No. 26703) on slant agar, a loopful of activated yeast cells was inoculated into 250 mL of liquid seed culture medium and cultured at 30 °C and 180 r / min on a shaker for 12 h. Subsequently, a 5% inoculum was inoculated into 2000 mL of fermentation medium and cultured at 30 °C and 180 r / min on a shaker for 24 h. After centrifugation, washing, and collection of the precipitated cells, the yeast cells were collected.

[0034] Example 2: Preparation of yeast protein products The yeast cells obtained from fermentation in Example 1 were diluted with water to prepare a 15% (w / w) suspension. NaOH was added to adjust the pH to 8.5, and the mixture was kept at 80°C for 120 minutes. After the treatment, the mixture was centrifuged at 5000 rpm, and the precipitate was collected.

[0035] The precipitate was dissolved in water to form a suspension with a weight percentage of 12%, and then homogenized twice using a high-pressure homogenizer at a pressure of 1000 Bar.

[0036] The homogenized suspension was adjusted to pH 5.0 with citric acid, and then 50 U / mL glucanase and 50 U / mL cellulase were added for enzymatic hydrolysis at 50 °C for 4 h.

[0037] The above liquid was centrifuged at 5000 rpm to separate it. The precipitate was redispersed with the same volume of 50℃ hot water, stirred and washed, and then centrifuged at 5000 rpm. The washing was repeated twice, and the centrifuged precipitate was collected.

[0038] The precipitate was freeze-dried to obtain the yeast protein product (KP). Testing showed that the prepared yeast protein product had a protein content of 78%.

[0039] Example 3: Simulated intestinal hydrolysis and peptide structure identification (1) In vitro simulated digestion The gastrointestinal simulation experiment is divided into two stages: gastric digestion (G) and gastrointestinal digestion (GI).

[0040] In vitro simulated gastric digestion (G): 100 mL of simulated gastric juice (pH 1.5) was added to a 200 mL brown test tube, and 1000 mg of yeast protein product (KP) sample prepared in Example 2 was added. Simulated gastric juice digestion was carried out at a constant temperature of 37℃, 120 rpm, and in the dark. At 0, 1 h, and 2 h, an appropriate amount of digestion product was taken to inactivate the enzyme (100℃, 10 min), centrifuged (10000 rpm, 20 min), and the supernatant was lyophilized. The product after 2 h of inactivation (i.e., digestion was completed) was used as the sample for subsequent experiments and was denoted as G-KP.

[0041] In vitro simulated intestinal digestion (GI): After adjusting the pH of the gastric digestion product G-KP to 6.8 with NaOH, 100 mL of simulated intestinal fluid (pH 6.8) was added. Simulated intestinal digestion was carried out at a constant temperature of 37℃ and 120 rpm in the dark. At 0, 1 h, and 2 h, an appropriate amount of digestion product was taken to inactivate the enzyme (100℃, 10 min), centrifuged (10000 rpm, 20 min), and the supernatant was lyophilized. The product after 2 h of inactivation (i.e., digestion was completed) was used as the sample for subsequent experiments and was denoted as GI-KP.

[0042] (2) Identification of polypeptide structure of digestion product A yeast protein gastrointestinal simulated digestion sample, GI-KP, was dissolved in mobile phase A (0.1% trifluoroacetic acid aqueous solution) to prepare a 0.5 mg / mL solution. One mL of this solution was desalted using a C18 column, filtered through a 0.22 µm microporous membrane, and then analyzed for peptide structure using nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS). The detection system consisted of an Orbitrap Exploris 480 coupled with an Easy-nLC 1200. 3 μL of sample was loaded onto a PepMap C18 (75 μm × 50 cm) column. The separation gradient was achieved by increasing mobile phase B (0.1% formic acid in acetonitrile) from 5% to 35% over 70 min. The chromatographic flow rate was 300 nL / min, the column temperature was 60 °C, and the ion source spray voltage was 2.3 kV.

[0043] The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z) 150-1600; resolution set to 120,000; maximum ion introduction time 50ms; automatic gain control (AGC) set to 5e5; (2) HCD-MS / MS (Top20): scan resolution set to 17500; minimum scan range fixed at m / z=110; maximum ion introduction time during MS / MS is 50ms; AGC control set to 2e5; precursor ion selection window set to 1.6 Da; for ions with charge numbers of 1, 2, 3, and 4, MS / MS acquisition was performed, and the dynamic exclusion time was 40s.

[0044] The raw data were processed and analyzed using PEAKS software. Peptide Ranker scoring was performed on 7039 peptides with an ALC (Average Local Confidence) score exceeding 95%, with a score threshold of 0.8, resulting in 128 peptides with high potential. These 128 peptides were then compared with DPP-4 inhibitory activities in the BIOPEP database, ultimately identifying 42 peptides with good DPP-4 inhibitory activity. The toxicity, solubility, intestinal absorption, and sensitization of the above 42 peptides were predicted using the ToxinPred, SwissADME, and Allergen FP databases. Finally, eight short peptides with excellent properties were screened out: FPSPM (Phe-Pro-Ser-Pro-Met), HFPF (His-Phe-Pro-Phe), FKEWL (Phe-Lys-Glu-Trp-Leu), AWLPR (Ala-Trp-Leu-Pro-Arg), GPAGPQGPR (Gly-Pro-Ala-Gly-Pro-Gln-Gly-Pro-Arg), GWV (Gly-Trp-Val), WLGGH (Trp-Leu-Gly-Gly-His), and LPFP (Leu-Pro-Phe-Pro). This indicates that these short peptide sequences are also present in yeast protein products (KP).

[0045] Example 4: Yeast protein digestion products promote GLP-1 secretion The main objective was to experimentally verify that yeast protein digestion products can promote the secretion of the incretin hormone glucagon-like peptide-1 (GLP-1).

[0046] (1) Culture of STC-1 cells (mouse small intestinal endocrine cells) STC-1 cells were cultured in DMEM medium supplemented with 10% FBS and then cultured to the logarithmic growth phase in a 37°C, 5% CO2 cell culture incubator before the experiment for subsequent experiments.

[0047] (2) Experiment on the cytotoxicity of yeast protein digestion products to STC-1 cells Cell viability was determined according to the procedure outlined in the MTT assay kit (Beyotime Biotechnology, C0009S). The effects of yeast protein gastrointestinal digestion products on STC-1 cell viability were assessed through cell proliferation and cytotoxicity assays. MTT was added to STC-1 cells after treatment in 96-well plates. Metabolically active cells lysed the yellow tetrazolium salt MTT into purple formazan crystals. The formed formazan was dissolved, and the absorbance was measured at 570 nm using a microplate reader. Results are expressed as a percentage of the control group. Specific methods were performed according to the manufacturer's instructions for the MTT cell proliferation and cytotoxicity assay kit.

[0048] (3) Effects of yeast protein digestion products on the secretion of GLP-1 and cholecystokinin (CCk) by STC-1 cells STC-1 cells were fed at a concentration of 1.25 × 10⁻⁶. 5 Cells were seeded at a density of 1,000 cells / well in 24-well plates. When the cells reached 80%–90% confluence, they were washed twice with Hank's buffer to remove the culture medium and incubated for 60 min in a buffer containing different concentrations of yeast protein gastrointestinal digestion products (Example 3). After incubation, the levels of GLP-1 and CCk secreted by STC-1 cells were measured according to the instructions of the GLP-1 and CCk assay kits (ELISA Biotechnology Co., Ltd., ml058435V-96T and ml201801V-96T).

[0049] from Figure 1 It can be seen that the yeast protein KP, yeast protein-mimicking intestinal digestion products G-KP and GI-KP obtained in Example 3 do not produce toxicity to STC-1 cells under the condition of 1-4 mg / mL.

[0050] from Figure 2 It can be seen that the yeast protein KP, yeast protein-mimicking intestinal digestion products G-KP, and GI-KP obtained in Example 3, at concentrations of 1-4 mg / mL, all significantly stimulated the secretion of GLP-1 and CCk. This indicates that the intact protein has a stronger stimulatory effect on CCk than the gastric and intestinal protein hydrolysates; while the gastric and intestinal protein hydrolysates have a stronger stimulatory effect on GLP-1 than the intact protein.

[0051] Example 5: Molecular docking simulates the binding of yeast protein digestion product peptides to DPP-4 Dipeptidyl peptidase-4 (DPP-4) can rapidly degrade glucagon-like peptide-1 (GLP-1), thereby reducing GLP-1's ability to induce β-cell differentiation and its stimulation of insulin synthesis and release. Inhibiting DPP-4 to prolong the half-life of GLP-1 in vivo is an important treatment method for diabetes. To further screen peptides with hypoglycemic activity, molecular docking technology was used to bind eight peptides identified in Example 3 to DPP-4, and active peptides were screened based on the binding site and binding energy.

[0052] The crystal structure of DPP-4 (PDB ID: 1TQF) with a resolution of 1.8 Å was obtained from the PDB database. Simultaneously, the three-dimensional structures of the peptides identified in Example 3 were plotted using ChemBio3D software. The structures of DPP-4 and the aforementioned peptides were imported into Autodock Vina 1.1.2 software. After dehydration and hydrogenation steps, molecular docking simulations were initiated. The results showed that three sequences had low binding energies to DPP-4: FPSPM, HFPF, and AWLPR. The mass spectra of these three peptides are shown below. Figure 3 , Figure 4 and Figure 5 As shown; global and detailed images of the three peptides docking with DPP-4 are shown in [image / image]. Figure 6 , Figure 7 and Figure 8 As shown.

[0053] The results showed that these three peptides (FPSPM, HFPF, and AWLPR) bound to DPP-4 via hydrogen bonds. The binding sites of FPSPM to DPP-4 were Ala306, Phe364, and Thr365, with a binding energy of -2.03 kcal / mol; the binding sites of HFPF to DPP-4 were Leu504, Gln505, and Val507, with a binding energy of -2.76 kcal / mol; and the binding site of AWLPR to DPP-4 was Ala743, with a binding energy of -1.7 kcal / mol. All formed relatively stable structures. Detailed data are shown in Table 1. This demonstrates that the FPSPM, HFPF, and AWLPR peptides have a good inhibitory effect on DPP-4, thereby reducing GLP-1 degradation and exhibiting a good function in lowering blood glucose.

[0054] Table 1. Binding of each peptide to DPP-4

[0055] Example 6: Kinetic analysis of the inhibition of DPP-4 by yeast protein digestion product peptides (1) Synthesis of polypeptides The three short peptides FPSPM (Phe-Pro-Ser-Pro-Met), HFPF (His-Phe-Pro-Phe), and AWLPR (Ala-Trp-Leu-Pro-Arg) were synthesized by Jier Biochemical (Shanghai) Co., Ltd.

[0056] (2) DPP-4 inhibitory activity assay The inhibitory activity of three peptides against DPP-4 was determined using a DPP-4 inhibitor activity assay kit (Elabscience, E-BC-D007). The experimental procedures were performed according to the kit instructions. The peptide concentrations (IC50) representing the half-maximal inhibitory rate were analyzed using Graphpad software. 50 (Value). Result as follows Figure 9 As shown, the IC50 of the peptide FPSPM 50 The concentration was 40.54 μg / mL; the IC50 of the peptide HFPF was... 50 The concentration was 36.97 μg / mL; the IC50 of the peptide AWLPR was... 50 The concentration was 53.71 μg / mL. This indicates that, based on inhibition kinetics, the three screened peptides all exhibited good inhibitory activity against DPP-4 at certain concentrations.

[0057] The above detailed description is a specific description of feasible embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. A yeast protein product with blood sugar lowering function, characterized in that, Its protein content is greater than 75%, and it contains specific amino acid sequences FPSPM, HFPF and AWLPR. FPSPM is shown in SEQ ID NO: 1, HFPF is shown in SEQ ID NO: 2 and AWLPR is shown in SEQ ID NO:

3.

2. A method for preparing a yeast protein product with blood sugar-lowering function as described in claim 1, characterized in that, Includes the following steps: S1. Yeast cell fermentation: After culturing yeast cells on slant culture medium, one loop of activated cells is inoculated into the planting medium and cultured on a shaker at 25-35℃ and 150-250 r / min for 12-24 h. Then, 5% of the inoculum is inoculated into the fermentation medium and cultured on a shaker at 25-35℃ and 150-250 r / min for 12-24 h. After centrifugation, washing, and centrifugation to collect the precipitate, the fermented yeast cells are obtained. S2. Heat treatment of yeast cells: Prepare a suspension of fermented yeast cells obtained in step S1 with a weight percentage of 5%-15%, adjust the pH to 7.0-9.0, set the temperature to 60-100℃, keep it at this temperature for 30-240 minutes, and then centrifuge to separate the precipitate. S3. High-pressure homogenization and cell disruption: Prepare an aqueous solution with a weight percentage of 2%-10% from the precipitate obtained in step S2, and homogenize and disrupt the cell walls 2-3 times using a high-pressure homogenizer at a pressure of 500-1500 Bar. S4. Enzymatic hydrolysis: Adjust the pH of the homogenized, cell-wall-broken solution obtained in step S3 to 5.0-7.0 and the temperature to 30-60℃. Add 5-50 U / mL of dextranase and 5-50 U / mL of cellulase and hydrolyze for 4-24 hours to obtain the enzymatic hydrolysate. S5. Solid-liquid separation: The enzymatic hydrolysate obtained in step S4 is subjected to solid-liquid separation, the precipitate is collected, and the precipitate is washed 2-3 times with the same volume of hot water at 40-60°C as the enzymatic hydrolysate. The precipitate is collected and dried to obtain the yeast protein product with the function of lowering blood sugar.

3. The method for preparing the yeast protein product with blood sugar lowering function according to claim 2, characterized in that, In step S1, the yeast is *Kluyveromyces*.

4. The method for preparing the yeast protein product with blood sugar lowering function according to claim 2, characterized in that, In step S1, the slant culture medium includes YPD medium and agar, wherein the weight percentage of the agar is 2%, and the YPD medium includes the following components in weight percentage: yeast extract 1%, peptone 2%, and glucose 2%. The seed culture medium includes YPD medium, which comprises the following components in weight percentage: yeast extract 1%, peptone 2%, and glucose 2%; The fermentation medium comprises the following components by weight percentage: glucose 6%, yeast extract 0.5%-1.0%, (NH4)2SO4 0.4%, KH2PO4 0.6%, K2SO4 0.1%, and the pH of the fermentation medium is 5.5; The slant culture medium, seed culture medium and fermentation culture medium were all sterilized under saturated steam at 121℃ for 15 min.

5. The method for preparing the yeast protein product with blood sugar lowering function according to claim 2, characterized in that, In step S2, the weight percentage of the prepared suspension is 6%-8%, the pH of the suspension is adjusted to 7.5-8.5, the holding temperature is 80-90℃, and the holding time is 60-120min.

6. The method for preparing the yeast protein product with blood sugar lowering function according to claim 2, characterized in that, In step S3, the weight percentage of the prepared aqueous solution is 5%-8%, and the pressure for cell disruption is 800-1200 Bar.

7. The method for preparing the yeast protein product with blood sugar lowering function according to claim 2, characterized in that, In step S4, the enzymatic hydrolysis temperature is 40-55℃ and the enzymatic hydrolysis time is 8-12h.

8. The method for preparing the yeast protein product with blood sugar lowering function according to claim 2, characterized in that, It also includes step S6, which involves identifying the amino acid sequence, and includes: S6-1. The yeast protein product is digested with simulated gastric juice to obtain product G-KP, and G-KP is then digested with simulated intestinal juice to obtain product GI-KP. S6-2 and GI-KP peptide structures were identified by nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS). The raw data were processed and analyzed using PEAKS software. After ALC scoring and DPP-4 inhibitory activity evaluation in the BIOPEP database, 42 peptides with good DPP-4 inhibitory activity were screened out. S6-3. The toxicity, solubility, intestinal absorption and sensitization of the 42 selected peptides were predicted using the ToxinPred, SwissADME and Allergen FP databases, respectively. Eight peptides with excellent properties were selected, namely FPSPM, HFPF, FKEWL, AWLPR, GPAGPQGPR, GWV, WLGGH and LPFP. S6-4. Using Autodock Vina software, the eight selected peptides were subjected to molecular docking simulation with DDP-4. Finally, three peptides with low binding energies to DPP-4 were selected, namely FPSPM, HFPF and AWLPR.

9. The method for preparing the yeast protein product with blood sugar lowering function according to claim 8, characterized in that, In step S6-2, the method for identifying the peptide structure of GI-KP using nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS) is as follows: GI-KP is dissolved in mobile phase A to prepare a 0.5 mg / mL solution. After desalting using a C18 column, the solution is filtered through a 0.22 µm microporous membrane and then injected into a NanoLC-MS / MS column. The column is a PepMap C18. Mobile phase A is a 0.1% trifluoroacetic acid aqueous solution, and mobile phase B is a 0.1% formic acid acetonitrile solution. The separation gradient is as follows: mobile phase B increases from 5% to 35% within 70 min. The sample loading volume is 3 μL, the chromatographic flow rate is 300 nL / min, and the column temperature is 60 °C. Mass spectrometry is performed using ion spray at a voltage of 2.3 kV. The method for selecting 42 peptides with good DPP-4 inhibitory activity after sequential ALC scoring and DPP-4 inhibitory activity evaluation in the BIOPEP database is as follows: the resolved peptides are scored using the Peptide Ranker score, with a score threshold of 0.8, and 128 peptides with high potential are selected; then the 128 selected peptides are compared with the DPP-4 inhibitory activity in the BIOPEP database, and finally 42 peptides with good DPP-4 inhibitory activity are selected.

10. The method for preparing the yeast protein product with blood sugar lowering function according to claim 8, characterized in that, In step S6-4, the method of using Autodock Vina software to simulate molecular docking between the eight selected peptides and DDP-4 is as follows: obtain the crystal structure of DPP-4 from the PDB database, and simultaneously use ChemBio3D software to draw the three-dimensional structure of each peptide to be screened; import the crystal structure of DPP-4 and the three-dimensional structure of the peptides into Autodock Vina software, and after dehydration and hydrogenation steps, start the molecular docking simulation, and screen the active peptides according to the strength of the binding sites and binding energies.

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