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CN-122011096-A - Yeast protein product with blood sugar reducing function and preparation method thereof

CN122011096ACN 122011096 ACN122011096 ACN 122011096ACN-122011096-A

Abstract

A yeast protein product with blood sugar reducing function is prepared through fermenting yeast, preparing the suspension of yeast, thermal insulating, collecting deposit, preparing 2-10% aqueous solution, homogenizing for breaking wall for 2-3 times under 500-1500Bar pressure, regulating pH, adding 5-50U/mL dextranase and 5-50U/mL cellulase, enzymolysis to obtain enzymolysis liquid, solid-liquid separation, washing, collecting deposit and drying. The yeast protein product prepared by the invention can promote the secretion of intestinal insulinotropic hormone such as GLP-1 and the like, inhibit DPP-4 activity to reduce the hydrolysis of GLP-1, has the effects of reducing blood sugar and weight, has no toxic or side effect, and has the advantages of simple preparation method, mild condition and suitability for mass production.

Inventors

  • ZHANG HAIBO
  • YAO ZHIJIAN
  • WANG ZHIMIN
  • PENG WENHUI

Assignees

  • 克鲁维酵母科技(海南)有限公司

Dates

Publication Date
20260512
Application Date
20260127

Claims (10)

  1. 1. A yeast protein product with the function of reducing blood sugar is characterized in that the protein content is more than 75%, and the yeast protein product contains specific amino acid sequences FPSPM, HFPF and AWPPR, wherein the FPSPM is shown as SEQ ID NO.1, HFPF is shown as SEQ ID NO. 2, and AWLPR is shown as SEQ ID NO. 3.
  2. 2. A method for preparing a yeast protein product having a blood sugar lowering function as claimed in claim 1, comprising the steps of: s1, fermenting yeast, namely culturing the yeast on a slant culture medium, inoculating a loop of activated yeast in a planting culture medium, performing shake culture for 12-24 hours at 25-35 ℃ and 150-250r/min, inoculating the yeast in a fermentation culture medium with an inoculum size of 5%, performing shake culture for 12-24 hours at 25-35 ℃ and 150-250r/min, performing centrifugal separation, washing, and centrifugally collecting sediment to obtain the fermented yeast; S2, heat treatment of yeast, namely preparing the fermented yeast obtained in the step S1 into a suspension with the weight percentage of 5-15%, regulating pH to 7.0-9.0, carrying out heat preservation treatment for 30-240min at 60-100 ℃, centrifuging, and collecting precipitate; s3, homogenizing and breaking the wall under high pressure, namely preparing the precipitate obtained in the step S2 into an aqueous solution with the weight percentage of 2-10%, and homogenizing and breaking the wall for 2-3 times under the pressure of 500-1500 Bar by adopting a high-pressure homogenizer; S4, enzyme hydrolysis, namely regulating the pH value of the solution obtained in the step 3 after homogenizing wall breaking to be 5.0-7.0, and adding 5-50U/mL of glucanase and 5-50U/mL of cellulase at the temperature of 30-60 ℃ for enzymolysis for 4-24 hours to obtain an enzymolysis solution; s5, solid-liquid separation, namely performing solid-liquid separation on the enzymolysis liquid obtained in the step S4, collecting the precipitate, washing the precipitate for 2-3 times by using 40-60 ℃ hot water with the same volume as the enzymolysis liquid, collecting the precipitate, and drying to obtain the yeast protein product with the blood sugar reducing function.
  3. 3. The method for producing a yeast protein product having a blood sugar lowering function according to claim 2, wherein in step S1, the yeast is kluyveromyces marxianus.
  4. 4. The method for preparing a yeast protein product with a blood sugar reducing function according to claim 2, wherein in the step S1, the slant culture medium comprises YPD culture medium and agar, the weight percentage of the agar is 2%, and the YPD culture medium comprises the following components by weight percentage of yeast extract 1%, peptone 2% and glucose 2%; the seed culture medium comprises YPD culture medium, wherein the YPD culture medium comprises the following components in percentage by weight of yeast extract 1%, peptone 2% and glucose 2%; the fermentation medium comprises the following components in percentage by weight of glucose 6%, and yeast extract 0.5%-1.0%,（NH 4 ） 2 SO 4 0.4%,KH 2 PO 4 0.6%,K 2 SO 4 0.1%, , wherein the pH of the fermentation medium is 5.5; The slant culture medium, the seed culture medium and the fermentation culture medium are sterilized for 15min under the saturated steam at 121 ℃.
  5. 5. The method for preparing a yeast protein product with a blood sugar reducing function according to claim 2, wherein in the 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 temperature is kept at 80-90 ℃, and the heat-preserving treatment time is 60-120min.
  6. 6. The method for preparing a yeast protein product with a blood sugar reducing function according to claim 2, wherein in the step S3, the weight percentage of the prepared aqueous solution is 5% -8%, and the wall breaking pressure is 800-1200Bar.
  7. 7. The method for preparing a yeast protein product with a blood sugar reducing function according to claim 2, wherein in the step S4, the enzymolysis temperature is 40-55 ℃ and the enzymolysis time is 8-12h.
  8. 8. The method for producing a yeast protein product having a blood sugar lowering function according to claim 2, further comprising an identification step S6 of the amino acid sequence, comprising: S6-1, digesting a yeast protein product by simulated gastric juice to obtain a product G-KP, and digesting the product G-KP by simulated intestinal juice to obtain a product GI-KP; s6-2 and GI-KP carry out polypeptide structure identification through nano liter liquid chromatography-tandem mass spectrometry NanoLC-MS/MS, the obtained original data are subjected to data processing and spectrogram analysis by PEAKS software, and 42 peptide fragments with better DPP-4 inhibitory activity are screened out after ALC scoring and DPP-4 inhibitory activity evaluation in BIOPEP database; S6-3, respectively predicting toxicity, solubility, intestinal absorbability and sensitization of the 42 peptide fragments by adopting ToxinPred, swissADME and ALLERGEN FP databases, respectively, and screening 8 peptide fragments with excellent characteristics, namely FPSPM, HFPF, FKEWL, AWLPR, GPAGPQGPR, GWV, WLGGH, LPFP; S6-4, molecular docking simulation is carried out on the 8 screened peptide fragments and DDP-4 by adopting Autodock vina software, and finally three peptide fragments with low binding energy with DPP-4 are screened out, namely FPSPM, HFPF and AWLPR.
  9. 9. The method for preparing the yeast protein product with the blood sugar reducing function according to claim 8, wherein in the step S6-2, the method for carrying out polypeptide structure identification on the GI-KP through nano liter liquid chromatography-tandem mass spectrometry NanoLC-MS/MS is characterized in that the GI-KP is dissolved in a mobile phase A to prepare a solution of 0.5mg/mL, then the solution is desalted by adopting a C18 small column, then the solution passes through a 0.22 mu m microporous filter membrane and enters into nano liter liquid chromatography-tandem mass spectrometry NanoLC-MS/MS, the chromatographic column is PepMap C, the chromatographic mobile phase A is 0.1% trifluoroacetic acid aqueous solution, the chromatographic mobile phase B is 0.1% formic acid acetonitrile solution, the separation gradient is 70 min, the mobile phase B rises from 5% to 35%, the loading amount is 3 mu L, the chromatographic flow rate is 300nL/min, the column temperature is 60 ℃, and the mass spectrometry adopts ion source spraying and the voltage is 2.3kV; The method for screening 42 peptide fragments with better DPP-4 inhibitory activity after ALC scoring and BIOPEP DPP-4 inhibitory activity evaluation in the database sequentially comprises the steps of scoring PEPTIDE RANKER of the analyzed peptide fragments, setting a score threshold value to be 0.8, screening 128 peptide fragments with high potential from the analyzed peptide fragments, comparing the screened 128 peptide fragments with the DPP-4 inhibitory activity in the BIOPEP database, and finally screening 42 peptide fragments with better DPP-4 inhibitory activity.
  10. 10. The method for preparing the yeast protein product with the blood sugar reducing function according to claim 8, wherein in the step S6-4, the method for performing molecular docking simulation on the screened 8 peptide fragments and DDP-4 respectively by adopting Autodock vina software is characterized in that the crystal structure of DPP-4 is obtained from a PDB database, meanwhile, the three-dimensional structure of each peptide fragment to be screened is drawn by using ChemBio D software, the crystal structure of DPP-4 and the three-dimensional structure of the peptide fragment are imported into Autodock vina software, and after dehydration and hydrogenation steps, molecular docking simulation is started, and active peptide fragments are screened according to the binding site and the strength of binding energy.

Description

Yeast protein product with blood sugar reducing function and preparation method thereof Technical Field The invention relates to the technical field of biological products, in particular to a yeast protein product with a blood sugar reducing function and a preparation method thereof. Background Diabetes is a metabolic disease that results in abnormally elevated blood glucose levels due to either insufficient insulin secretion or insulin resistance. Long-term hyperglycemia is prone to a variety of long-term health complications, including heart disease, stroke, systemic blood circulation disorders, and the like. The disease also presents a high-rise situation in China, and a research report issued by 2025, 6 and 2 days shows that the estimated rate of the diabetes mellitus patients in 2023 is 2.33 hundred million, and the age-standardized prevalence rate is 13.7%. At present, the drug treatment is one of the core means of diabetes management and is mainly divided into two major categories of oral hypoglycemic agents and injection hypoglycemic agents, wherein the injection medicaments comprise insulin and glucagon-like peptide-1 receptor agonist (GLP-1), and the oral hypoglycemic agents cover main categories such as biguanides, sulfonylureas, sodium-glucose cotransporter 2 inhibitors and the like. Obesity has become one of the major health challenges currently faced in china. Glucagon-like peptide-1 (Glucagon-likepeptide-1, glp-1) is an incretin and a hormone consisting of 37 amino acids, and has the core physiological functions of stimulating insulin secretion, protecting pancreatic beta cells, inhibiting glucagon secretion, delaying gastric emptying and reducing food intake, and finally realizing the dual effects of reducing blood sugar and reducing weight. However, GLP-1 secretion is impaired in diabetic and obese patients, and postprandial GLP-1 secretion is significantly reduced compared to normal populations, resulting in reduced postprandial glycemic control. Liraglutide (Liraglutide) and semraglutide (Semaglutide) have been batched for the treatment of type 2 diabetes and obesity as GLP-1 receptor agonists. However, natural GLP-1 is easily inactivated or degraded rapidly by enzymes such as dipeptidyl peptidase-4 (DPP-4), and the half-life is only about 1.5 minutes, which severely limits its blood glucose regulating efficacy. DPP-4 is a serine peptidase on the cell surface, belongs to the membrane protein family, is widely distributed in kidney, connective tissue, gastrointestinal tract, lymph node and other tissues, and can rapidly degrade GLP-1 and other incretin. Therefore, the food component with the functions of promoting GLP-1 secretion or inhibiting DPP-4 activity is developed, and the functions of reducing blood sugar and improving body weight are better. The long-acting GLP-1 derivative and the GLP-1 receptor agonist have better secretin effect, so that the long-acting GLP-1 derivative and the GLP-1 receptor agonist become important research points in the field of diabetes control, and the GLP-1 analogue and the receptor agonist are widely known in the prior art. GLP-1 belongs to an incretin hormone, and receptor signaling of GLP-1 has important physiological significance for maintaining glucose homeostasis. In the prior art, CN119212574A discloses a hydrolyzed collagen preparation with hypoglycemic effect, which is prepared by carrying out enzymatic hydrolysis on raw materials containing collagen through protease combination, is suitable for being used as a dietary supplement to assist in improving hyperglycemia state and related risk factors, and JP2012116773 discloses a collagen peptide derived from fish scale and/or fish skin collagen, which is prepared through protease treatment and can improve Insulin-like growth factor1 (Insulin-likegrowthfactor, IGF-1) level. CN 202210528272.3 discloses a DPP-4 inhibitory peptide derived from yak collagen, the sequence is MGPR, and the DPP-4 inhibitory activity is achieved. Although the prior art has developed a variety of active incretin analogues, there are a number of limitations to the use of such products as pharmaceuticals. Therefore, the development of a product which has definite activity and better safety and is capable of acting on incretins and is a food source, and the provision of safer and more effective auxiliary foods for obesity, diabetes and complications thereof has become an important subject to be solved in the field. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a yeast protein product with a blood sugar reducing function and a preparation method thereof, wherein the yeast protein product contains specific amino acid sequences which can obviously promote secretion of glucagon-like peptide (GLP-1) in gastrointestinal digestion products, inhibit DPP-4 activity to reduce decomposition of GLP-1, and have the effects of reducing blood sugar and improving body weight. The invention is re