Food protein peptide with hypoglycemic activity and preparation method thereof

Abstract

This invention belongs to the field of functional food technology, specifically relating to a food protein peptide with hypoglycemic activity and its preparation method. This invention uses gallic acid-modified yam protein peptides, which synergistically enhance the inhibition rates of α-amylase and α-glucosidase in the food protein peptides, improving hypoglycemic efficiency. The phenolic structure contained in gallic acid also possesses antioxidant properties, maintaining the active conformation of the protein peptides and improving their oxidative stability. The gallic acid-modified yam protein peptides can scavenge excess free radicals in the high-glycemic environment of diabetic patients, reducing damage to pancreatic β-cells and helping to alleviate glucose metabolism damage. The gallic acid-modified yam protein peptides are encapsulated using chitosan-STPP as a nanocarrier; its electrostatic cross-linked network structure can resist the degradation of the gallic acid-modified yam protein peptides by pepsin, ensuring the hypoglycemic activity of the food protein peptides. After entering the alkaline environment of the intestine, the carrier swells and loosens, slowly releasing the active ingredients, thus stabilizing blood glucose fluctuations.

Description

Technical Field

[0001] This invention belongs to the field of functional food technology, specifically relating to a food protein peptide with hypoglycemic activity and its preparation method. Background Technology

[0002] Diabetes is currently the third leading cause of death from COVID-19 among non-communicable diseases, after cancer and cardiovascular diseases. It is a difficult-to-cure disease with a high incidence rate that is increasing annually, making it a significant global health problem. The typical metabolic characteristics of diabetic patients are disorders of glucose and lipid metabolism. It is divided into two types: type 1 diabetes and type 2 diabetes. Type 1 diabetes is caused by the destruction of pancreatic islets by the autoimmune system, leading to the loss of insulin secretion function and loss of glucose metabolism control. Type 2 diabetes involves multiple factors that can alter glucose metabolism and uptake pathways in various organs and tissues, such as the liver, muscles, and adipose tissue. In addition, another important characteristic of type 2 diabetes is insulin resistance, which mainly refers to the disruption of the insulin signaling mechanism, leading to impaired energy metabolism.

[0003] Currently, biological and chemical drugs remain the primary treatments for diabetes. Insulin and metformin are the first-line drugs for type 1 and type 2 diabetes, respectively, primarily acting through the insulin-dependent pathway. Combined with other measures such as regulating glucose and lipid metabolism and controlling blood pressure, these drugs have helped manage the condition of countless diabetic patients. However, in the later stages of the disease, patients experience increased insulin resistance, leading to a greater demand for insulin. This increased demand further burdens the pancreas, causing further damage. Lowering blood sugar in diabetic patients through non-insulin-dependent pathways not only reduces the burden on the pancreas but can also work synergistically with insulin-dependent hypoglycemic products.

[0004] Yam is a food with both medicinal and nutritional value, possessing effects such as immune regulation, anti-oxidation, anti-aging, and regulation of blood lipids, blood sugar, and acid-base balance. Currently, the active ingredients in yam-based drugs and raw materials that have a hypoglycemic effect are relatively simple, and their effect on lowering blood sugar is very limited, and they still cannot completely replace traditional synthetic chemical drugs for lowering blood sugar. Summary of the Invention

[0005] The purpose of this invention is to provide a food protein peptide with hypoglycemic activity and its preparation method, so as to solve the above-mentioned technical problems.

[0006] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: A method for preparing a food protein peptide with hypoglycemic activity includes the following steps: S1. Take solution A and solution B at a volume ratio of 5~7:1. Add solution B dropwise to solution A at a rate of 0.5~1.0 mL / min with stirring. After the addition is complete, continue stirring for 2 hours. Then centrifuge at 4℃ and 8000 rpm. After centrifugation, discard the supernatant. Wash the precipitate with deionized water and centrifuge to obtain the solid phase. Resuspend the obtained solid phase with deionized water at a solid-liquid ratio of 1 g:10 mL to obtain a suspension. S2. Add mannitol to the suspension, mix thoroughly, pre-freeze at -80℃, and then freeze-dry at -50℃ and 10kPa to obtain food protein peptides with hypoglycemic activity; the amount of mannitol added is 5-8 wt% of the solid phase contained in the suspension. The A solution is composed of 1.5-2.0% acetic acid solution, chitosan, EDTA-2Na, and gallic acid-modified yam protein peptide powder. Solution B is a STPP solution with a mass concentration of 0.1~0.3%.

[0007] As a further improvement, the preparation method of solution A is as follows: using 1.5~2.0% acetic acid solution as solvent, chitosan is added under stirring to prepare a chitosan solution with a concentration of 1.0~2.0 mg / mL. EDTA-2Na is added under stirring and completely dissolved. Gallic acid modified yam protein peptide powder is added and the pH is adjusted to 4.4~4.6. After filtration through a filter membrane, solution A is obtained and placed at room temperature for later use.

[0008] As a further improvement, the preparation method of the gallic acid-modified yam protein peptide powder is as follows: S1. Under water bath heating at 45~50℃, add the pretreated yam protein powder to deionized water at a mass ratio of 1:15, adjust the pH to 6.5~6.9, and after enzymatic hydrolysis by neutral protease and flavor protease, raise the temperature to 85℃ and keep it constant for 10~15 minutes to inactivate the enzymes. S2. After inactivation, the product undergoes two-stage coarse separation followed by fine separation, then pre-concentration via reverse osmosis membrane, and finally vacuum membrane concentration at 55-60℃ for 3-5 times. After concentration, the product is spray-dried to prepare peptide powder, yielding yam protein peptide powder. S3. Add yam protein peptide powder to phosphate buffer solution with pH 9 at a solid-liquid ratio of 1:100. Stir until completely dissolved, and then add gallic acid diluted with anhydrous ethanol under light-protected conditions. Stir and react at 30°C for 14-16 hours. After the reaction is complete, centrifuge and take the supernatant. After dialyzing and freeze-drying, the obtained supernatant is used to obtain gallic acid modified yam protein peptide powder.

[0009] As a further improvement, the pretreatment method of the yam protein powder is as follows: dissolve the yam protein powder in deionized water at a mass ratio of 1:10, adjust the pH to 6.5~7.0, add α-amylase, react at 60℃ for 30 min, and after the reaction is completed, centrifuge, wash, and vacuum dry to obtain the pretreated yam protein powder; the amount of α-amylase added is 0.3~0.5 wt% of the yam protein powder.

[0010] As a further improvement, in step S1, the amount of neutral protease added is 1.5~2.0 wt% of the pretreated yam protein powder; the amount of flavor protease added is 1.0~1.5 wt% of the pretreated yam protein powder.

[0011] As a further improvement, in step S2, the two-stage coarse separation method is as follows: first, preliminary filtration is completed through a 200-mesh sieve, and then microfiltration is performed using a 0.2μm ceramic membrane; the fine separation method is as follows: at 0.2MPa, 25℃, and a flow rate of 2~3m / s, the liquid is first filtered through a 10kDa ultrafiltration membrane, the permeate is collected, and then the obtained permeate is filtered through a 1kDa ultrafiltration membrane, the retentate is collected, and the fine separation is completed.

[0012] As a further improvement, in step S3, the amount of gallic acid added is 7.5~12.5wt% of the yam protein peptide powder; the concentration of gallic acid after dilution with anhydrous ethanol is 50~60mg / mL.

[0013] As a further improvement, the amount of gallic acid-modified yam protein peptide powder added is 10-30 wt% of chitosan; the mass ratio of chitosan to EDTA-2Na is 1:1.

[0014] As a further improvement, the preparation method of the B solution is as follows: weigh STPP, dissolve it in deionized water and stir evenly to prepare a STPP solution with a mass concentration of 0.1~0.3%, filter it through a 0.22μm filter membrane to obtain the B solution, and seal and store it for later use.

[0015] The present invention also provides a food protein peptide with hypoglycemic activity.

[0016] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: 1. Gallic acid is used to modify yam protein peptides. The two work synergistically to enhance the inhibition rate of α-amylase and α-glucosidase, thereby improving the hypoglycemic efficiency of food protein peptides. The phenolic structure in gallic acid has antioxidant activity, which can protect yam protein peptides from oxidation, maintain their active conformation, improve the oxidative stability of food protein peptides, and facilitate the exertion of hypoglycemic activity.

[0017] 2. Gallic acid and yam protein peptides work synergistically to eliminate a large number of free radicals generated in the body of diabetic patients under high sugar conditions, reduce the attack of free radicals on pancreatic β cells, improve the survival rate of pancreatic β cells, and help reduce the degree of damage from glucose metabolism.

[0018] 3. Chitosan-STPP is used as a nanocarrier to encapsulate gallic acid-modified yam protein peptides. Chitosan and STPP form a network structure through electrostatic interaction, creating a physical barrier that reduces the degradation of gallic acid-modified yam protein peptides by pepsin, thus ensuring the hypoglycemic activity of the food protein peptides. After the food protein peptides enter the alkaline environment of the intestine, the protonation degree of chitosan decreases, the electrostatic cross-linking with STPP weakens, the carrier structure swells and loosens, and the gallic acid-modified yam protein peptides are slowly released, achieving the continuous release of hypoglycemic active ingredients and helping to maintain stable blood sugar levels. Attached Figure Description

[0019] Figure 1 Infrared spectra of yam protein peptide powder before and after modification with gallic acid. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or manufacturer's conditions are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. The yam protein powder used is commercially available defatted yam protein powder from Anyang Tianxiangrui Food Technology Co., Ltd., with a protein content of 52.2%.

[0021] Example 1: A method for preparing a food protein peptide with hypoglycemic activity, specifically including the following steps: 1. Dissolve yam protein powder in deionized water at a mass ratio of 1:10, adjust the pH to 6.5 using citrate-sodium citrate buffer, add α-amylase, react at 60℃ for 30 min, after the reaction is complete, centrifuge at 8000 rpm for 10 min, collect the solid phase, wash 3 times with deionized water, and vacuum dry at 55℃ to constant weight to obtain pretreated yam protein powder; the amount of α-amylase added is 0.3 wt% of yam protein powder.

[0022] 2. Under 45℃ water bath heating, add the pretreated yam protein powder to deionized water at a mass ratio of 1:15. Adjust the pH using citrate-sodium citrate buffer, add neutral protease, and continue enzymatic hydrolysis at 50℃ and pH 6.8 for 2 hours. Then add flavor protease and continue enzymatic hydrolysis at 50℃ and pH 6.5 for 2 hours. After enzymatic hydrolysis, raise the temperature to 85℃ and maintain the temperature for 10 minutes to inactivate the enzyme. The amount of neutral protease added is 1.5 wt% of the pretreated yam protein powder, and the amount of flavor protease added is 1.0 wt% of the pretreated yam protein powder.

[0023] 3. After inactivation, two-stage coarse separation is performed: First, preliminary filtration is completed through a 200-mesh sieve, and then microfiltration is performed using a 0.2μm ceramic membrane to remove macromolecular impurities and insoluble residues; after coarse separation, fine separation is performed: at 0.2MPa, 25℃, and a flow rate of 2m / s, the solution is first filtered through a 10kDa ultrafiltration membrane, the permeate is collected, and unhydrolyzed macromolecular proteins and peptides above 10kDa in the system are removed. Then, the obtained permeate is filtered through a 1kDa ultrafiltration membrane, the retentate is collected, and fine separation is completed.

[0024] 4. After fine separation, the retentate was pre-concentrated using a reverse osmosis membrane to a volume of 1 / 4. Then, vacuum membrane concentration was performed at 55℃ and 0.05MPa. After three concentrations, peptide powder was prepared by spray drying. The inlet air temperature was set to 180℃ and the outlet air temperature to 85℃. After spray drying, yam protein peptide powder was obtained. The amino acid composition of the yam protein peptide powder was determined using an automatic amino acid analyzer. The results are shown in Table 1. Table 1. Amino acid composition analysis of yam protein peptide powder

[0025] 5. Add yam protein peptide powder to phosphate buffer solution with pH 9 at a solid-liquid ratio of 1g:100mL, stir at 400rpm for 30min, add gallic acid diluted with anhydrous ethanol under light-protected conditions, stir at 500rpm for 14h at 30℃, after the reaction is complete, centrifuge at 8000rpm for 10min at 4℃, and take the supernatant; the amount of gallic acid added is 7.5wt% of yam protein peptide powder; the concentration of gallic acid after dilution with anhydrous ethanol is 50mg / mL.

[0026] 6. Using a dialysis bag with a molecular weight cutoff of 1 kDa, dialyze the supernatant obtained in step 5 at 4°C for 24 hours, changing the ultrapure water every 6 hours. Collect the retentate, freeze-dry it at -30°C to obtain gallic acid-modified yam protein peptide powder. Infrared spectroscopy was performed on the yam protein peptide powder before and after gallic acid modification. The test results are as follows: Figure 1As shown, a represents the infrared spectrum of yam protein peptide powder before modification, and b represents the infrared spectrum of yam protein peptide powder after gallic acid modification. In the infrared spectrum of the modified yam protein peptide powder, the value at 1750 cm⁻¹... -1 The C=O stretching vibration peak of carboxylic acid was significantly enhanced, and the peak at 750 cm⁻¹ was also significantly enhanced. -1 The presence of characteristic peaks of ortho-substituted benzene rings nearby proves that gallic acid has been successfully grafted onto yam protein peptide powder.

[0027] 7. Using a 1.5% acetic acid solution as a solvent, chitosan was added with stirring to prepare a chitosan solution with a concentration of 1.0 mg / mL. Disodium ethylenediaminetetraacetate (EDTA-2Na) was added with stirring until completely dissolved. Gallic acid-modified yam protein peptide powder was then added. The pH was adjusted to 4.4 using 0.1 mol / L NaOH solution. The solution was filtered through a 0.22 μm filter membrane to obtain solution A, which was then stored at room temperature for later use. The amount of gallic acid-modified yam protein peptide powder added was 10 wt% of the chitosan; the mass ratio of chitosan to EDTA-2Na was 1:1. Weigh out sodium tripolyphosphate (STPP), dissolve it in deionized water and stir until homogeneous to prepare a 0.1% STPP solution. Filter the solution through a 0.22μm filter membrane to obtain solution B, which should be sealed and stored for use.

[0028] 8. Take solution A and solution B at a volume ratio of 5:1. While stirring at 800 rpm, add solution B dropwise to solution A at a rate of 0.5 mL / min, stirring continuously during the addition. After the addition is complete, continue stirring for 2 hours. Centrifuge at 4℃ and 8000 rpm for 30 minutes. After centrifugation, discard the supernatant and wash the precipitate with deionized water. After washing, centrifuge again and discard the supernatant to obtain the solid phase. Resuspend the obtained solid phase with deionized water at a solid-liquid ratio of 1 g: 10 mL to obtain a suspension.

[0029] 9. Add mannitol to the suspension as a freeze-drying protectant, mix thoroughly, pre-freeze at -80℃ for 0.8h, and then freeze-dry at -50℃ and 10kPa for 8h to obtain food protein peptides with hypoglycemic activity; the amount of mannitol added is 5wt% of the solid phase contained in the suspension.

[0030] Example 2: A method for preparing a food protein peptide with hypoglycemic activity, specifically including the following steps: 1. Dissolve yam protein powder in deionized water at a mass ratio of 1:10, adjust the pH to 6.8 using citrate-sodium citrate buffer, add α-amylase, react at 60℃ for 30 min, after the reaction is complete, centrifuge at 8000 rpm for 10 min, collect the solid phase, wash 3 times with deionized water, and vacuum dry at 55℃ to constant weight to obtain pretreated yam protein powder; the amount of α-amylase added is 0.4 wt% of yam protein powder.

[0031] 2. Under 48℃ water bath heating, the pretreated yam protein powder was added to deionized water at a mass ratio of 1:15. The pH was adjusted using citrate-sodium citrate buffer, and neutral protease was added. Enzymatic hydrolysis was continued at 50℃ and pH 6.8 for 2 hours. Then, flavor protease was added, and enzymatic hydrolysis was continued at 50℃ and pH 6.5 for 2 hours. After enzymatic hydrolysis, the temperature was raised to 85℃ and held for 10 minutes to inactivate the enzyme. The amount of neutral protease added was 1.8 wt% of the pretreated yam protein powder, and the amount of flavor protease added was 1.3 wt% of the pretreated yam protein powder.

[0032] 3. After inactivation, two-stage coarse separation is performed: First, preliminary filtration is completed through a 200-mesh sieve, and then microfiltration is performed using a 0.2μm ceramic membrane to remove macromolecular impurities and insoluble residues; after coarse separation, fine separation is performed: at 0.2MPa, 25℃, and a flow rate of 3m / s, the solution is first filtered through a 10kDa ultrafiltration membrane, the permeate is collected, and unhydrolyzed macromolecular proteins and peptides above 10kDa in the system are removed. Then, the obtained permeate is filtered through a 1kDa ultrafiltration membrane, the retentate is collected, and fine separation is completed.

[0033] 4. After fine separation, the retentate is pre-concentrated using a reverse osmosis membrane. After the volume of the concentrated juice is reduced to 1 / 5, vacuum membrane concentration is carried out at 58℃ and 0.07MPa. After concentration four times, peptide powder is prepared by spray drying. The inlet air temperature is set to 180℃ and the outlet air temperature is set to 85℃. After spray drying, yam protein peptide powder is obtained.

[0034] 5. Add yam protein peptide powder to phosphate buffer solution with pH 9 at a solid-liquid ratio of 1g:100mL, stir at 500rpm for 45min, add gallic acid diluted with anhydrous ethanol under light-protected conditions, stir at 550rpm for 15h at 30℃, after the reaction is complete, centrifuge at 8000rpm for 10min at 4℃, and take the supernatant; the amount of gallic acid added is 10wt% of yam protein peptide powder; the concentration of gallic acid after dilution with anhydrous ethanol is 55mg / mL.

[0035] 6. Using a dialysis bag with a molecular weight cutoff of 1 kDa, dialyze the supernatant obtained in step 5 at 4°C for 24 hours, changing the ultrapure water every 6 hours. Collect the retentate and freeze-dry it at -30°C to obtain gallic acid modified yam protein peptide powder.

[0036] 7. Using a 1.8% acetic acid solution as a solvent, chitosan was added with stirring to prepare a chitosan solution with a concentration of 1.5 mg / mL. EDTA-2Na was added with stirring until completely dissolved. Gallic acid-modified yam protein peptide powder was then added. The pH was adjusted to 4.5 using 0.1 mol / L NaOH solution. The solution was filtered through a 0.22 μm filter membrane to obtain solution A, which was then stored at room temperature for later use. The amount of gallic acid-modified yam protein peptide powder added was 20 wt% of the chitosan; the mass ratio of chitosan to EDTA-2Na was 1:1. Weigh out STPP, dissolve it in deionized water and stir until homogeneous to prepare a 0.2% STPP solution. Filter the solution through a 0.22μm filter membrane to obtain solution B, and store it in a sealed container for use.

[0037] 8. Take solution A and solution B at a volume ratio of 6:1. While stirring at 800 rpm, add solution B dropwise to solution A at a rate of 0.8 mL / min, stirring continuously during the addition. After the addition is complete, continue stirring for 2 hours. Centrifuge at 4℃ and 8000 rpm for 30 minutes. After centrifugation, discard the supernatant and wash the precipitate with deionized water. After washing, centrifuge again and discard the supernatant to obtain the solid phase. Resuspend the obtained solid phase with deionized water at a solid-liquid ratio of 1 g: 10 mL to obtain a suspension.

[0038] 9. Add mannitol to the suspension as a freeze-drying protectant, mix thoroughly, pre-freeze at -80℃ for 0.9h, and then freeze-dry at -50℃ and 10kPa for 8h to obtain food protein peptides with hypoglycemic activity; the amount of mannitol added is 7wt% of the solid phase contained in the suspension.

[0039] Example 3: A method for preparing a food protein peptide with hypoglycemic activity, specifically including the following steps: 1. Dissolve yam protein powder in deionized water at a mass ratio of 1:10, adjust the pH to 7.0 using citrate-sodium citrate buffer, add α-amylase, react at 60℃ for 30 min, after the reaction is complete, centrifuge at 8000 rpm for 10 min, collect the solid phase, wash 3 times with deionized water, and vacuum dry at 55℃ to constant weight to obtain pretreated yam protein powder; the amount of α-amylase added is 0.5 wt% of yam protein powder.

[0040] 2. Under 50℃ water bath heating, add the pretreated yam protein powder to deionized water at a mass ratio of 1:15. Adjust the pH using citrate-sodium citrate buffer, add neutral protease, and continue enzymatic hydrolysis at 50℃ and pH 6.8 for 2 hours. Then add flavor protease and continue enzymatic hydrolysis at 50℃ and pH 6.5 for 2 hours. After enzymatic hydrolysis, raise the temperature to 85℃ and maintain the temperature for 10 minutes to inactivate the enzyme. The amount of neutral protease added is 2.0 wt% of the pretreated yam protein powder, and the amount of flavor protease added is 1.5 wt% of the pretreated yam protein powder.

[0041] 3. After inactivation, two-stage coarse separation is performed: First, preliminary filtration is completed through a 200-mesh sieve, and then microfiltration is performed using a 0.2μm ceramic membrane to remove macromolecular impurities and insoluble residues; after coarse separation, fine separation is performed: at 0.2MPa, 25℃, and a flow rate of 3m / s, the solution is first filtered through a 10kDa ultrafiltration membrane, the permeate is collected, and unhydrolyzed macromolecular proteins and peptides above 10kDa in the system are removed. Then, the obtained permeate is filtered through a 1kDa ultrafiltration membrane, the retentate is collected, and fine separation is completed.

[0042] 4. After fine separation, the retentate is pre-concentrated using a reverse osmosis membrane. After concentration to 1 / 6 of the retentate volume, vacuum membrane concentration is carried out at 60℃ and 0.09MPa. After concentration five times, peptide powder is prepared by spray drying. The inlet air temperature is set to 180℃ and the outlet air temperature is set to 85℃. After spray drying, yam protein peptide powder is obtained.

[0043] 5. Add yam protein peptide powder to phosphate buffer solution with pH 9 at a solid-liquid ratio of 1g:100mL, stir at 600rpm for 60min, add gallic acid diluted with anhydrous ethanol under light-protected conditions, stir at 600rpm for 16h at 30℃, after the reaction is complete, centrifuge at 8000rpm for 10min at 4℃, and take the supernatant; the amount of gallic acid added is 12.5wt% of yam protein peptide powder; the concentration of gallic acid after dilution with anhydrous ethanol is 60mg / mL.

[0044] 6. Using a dialysis bag with a molecular weight cutoff of 1 kDa, dialyze the supernatant obtained in step 5 at 4°C for 24 hours, changing the ultrapure water every 6 hours. Collect the retentate and freeze-dry it at -30°C to obtain gallic acid modified yam protein peptide powder.

[0045] 7. Using a 2.0% acetic acid solution as a solvent, chitosan was added with stirring to prepare a chitosan solution with a concentration of 2.0 mg / mL. EDTA-2Na was added with stirring until completely dissolved. Gallic acid-modified yam protein peptide powder was then added. The pH was adjusted to 4.6 using 0.1 mol / L NaOH solution. The solution was filtered through a 0.22 μm filter membrane to obtain solution A, which was then stored at room temperature for later use. The amount of gallic acid-modified yam protein peptide powder added was 30 wt% of the chitosan; the mass ratio of chitosan to EDTA-2Na was 1:1. Weigh out STPP, dissolve it in deionized water and stir until homogeneous to prepare a 0.3% STPP solution. Filter the solution through a 0.22μm filter membrane to obtain solution B, and store it in a sealed container for use.

[0046] 8. Take solution A and solution B at a volume ratio of 7:1. While stirring at 800 rpm, add solution B dropwise to solution A at a rate of 1.0 mL / min, stirring continuously during the addition. After the addition is complete, continue stirring for 2 hours. Centrifuge at 4℃ and 8000 rpm for 30 minutes. After centrifugation, discard the supernatant and wash the precipitate with deionized water. After washing, centrifuge again and discard the supernatant to obtain the solid phase. Resuspend the obtained solid phase with deionized water at a solid-liquid ratio of 1 g: 10 mL to obtain a suspension.

[0047] 9. Add mannitol to the suspension as a freeze-drying protectant, mix thoroughly, pre-freeze at -80℃ for 1 hour, and then freeze-dry at -50℃ and 10kPa for 8 hours to obtain food protein peptides with hypoglycemic activity; the amount of mannitol added is 8wt% of the solid phase contained in the suspension.

[0048] Comparative Example 1: A method for preparing a food protein peptide with hypoglycemic activity, differing from Example 1 in that the yam protein peptide is not modified with gallic acid, specifically including the following steps: 1. Dissolve yam protein powder in deionized water at a mass ratio of 1:10, adjust the pH to 6.5 using citrate-sodium citrate buffer, add α-amylase, react at 60℃ for 30 min, after the reaction is complete, centrifuge at 8000 rpm for 10 min, collect the solid phase, wash 3 times with deionized water, and vacuum dry at 55℃ to constant weight to obtain pretreated yam protein powder; the amount of α-amylase added is 0.3 wt% of yam protein powder.

[0049] 2. Under 45℃ water bath heating, add the pretreated yam protein powder to deionized water at a mass ratio of 1:15. Adjust the pH using citrate-sodium citrate buffer, add neutral protease, and continue enzymatic hydrolysis at 50℃ and pH 6.8 for 2 hours. Then add flavor protease and continue enzymatic hydrolysis at 50℃ and pH 6.5 for 2 hours. After enzymatic hydrolysis, raise the temperature to 85℃ and maintain the temperature for 10 minutes to inactivate the enzyme. The amount of neutral protease added is 1.5 wt% of the pretreated yam protein powder, and the amount of flavor protease added is 1.0 wt% of the pretreated yam protein powder.

[0050] 3. After inactivation, two-stage coarse separation is performed: First, preliminary filtration is completed through a 200-mesh sieve, and then microfiltration is performed using a 0.2μm ceramic membrane to remove macromolecular impurities and insoluble residues; after coarse separation, fine separation is performed: at 0.2MPa, 25℃, and a flow rate of 2m / s, the solution is first filtered through a 10kDa ultrafiltration membrane, the permeate is collected, and unhydrolyzed macromolecular proteins and peptides above 10kDa in the system are removed. Then, the obtained permeate is filtered through a 1kDa ultrafiltration membrane, the retentate is collected, and fine separation is completed.

[0051] 4. After fine separation, the retentate is pre-concentrated using a reverse osmosis membrane. After concentration to 1 / 4 of the retentate volume, vacuum membrane concentration is carried out at 55℃ and 0.05MPa. After concentration three times, peptide powder is prepared by spray drying. The inlet air temperature is set to 180℃ and the outlet air temperature is set to 85℃. After spray drying, yam protein peptide powder is obtained.

[0052] 5. Using a 1.5% acetic acid solution as a solvent, chitosan was added with stirring to prepare a chitosan solution with a concentration of 1.0 mg / mL. EDTA-2Na was added with stirring until completely dissolved. Then, yam protein peptide powder was added, and the pH was adjusted to 4.4 using 0.1 mol / L NaOH solution. The solution was filtered through a 0.22 μm filter membrane to obtain solution A, which was then stored at room temperature for later use. The amount of yam protein peptide powder added was 10 wt% of the chitosan; the mass ratio of chitosan to EDTA-2Na was 1:1. Weigh out STPP, dissolve it in deionized water and stir until homogeneous to prepare a 0.1% STPP solution. Filter the solution through a 0.22μm filter membrane to obtain solution B, and store it in a sealed container for use.

[0053] 6. Take solution A and solution B at a volume ratio of 5:1. While stirring at 800 rpm, add solution B dropwise to solution A at a rate of 0.5 mL / min, stirring continuously during the addition. After the addition is complete, continue stirring for 2 hours. Centrifuge at 4℃ and 8000 rpm for 30 minutes. After centrifugation, discard the supernatant and wash the precipitate with deionized water. After washing, centrifuge again and discard the supernatant to obtain the solid phase. Resuspend the obtained solid phase with deionized water at a solid-liquid ratio of 1 g: 10 mL to obtain a suspension.

[0054] 7. Add mannitol to the suspension as a freeze-drying protectant, mix thoroughly, pre-freeze the sample at -80℃ for 0.8h, and then freeze-dry at -50℃ and 10kPa for 8h to obtain food protein peptides with hypoglycemic activity; the amount of mannitol added is 5wt% of the solid phase contained in the suspension.

[0055] Comparative Example 2: A method for preparing a food protein peptide with hypoglycemic activity, differing from Example 1 in that it does not use chitosan-STPP nanocarriers, and specifically includes the following steps: 1. Dissolve yam protein powder in deionized water at a mass ratio of 1:10, adjust the pH to 6.5 using citrate-sodium citrate buffer, add α-amylase, react at 60℃ for 30 min, after the reaction is complete, centrifuge at 8000 rpm for 10 min, collect the solid phase, wash 3 times with deionized water, and vacuum dry at 55℃ to constant weight to obtain pretreated yam protein powder; the amount of α-amylase added is 0.3 wt% of yam protein powder.

[0056] 2. Under 45℃ water bath heating, add the pretreated yam protein powder to deionized water at a mass ratio of 1:15. Adjust the pH using citrate-sodium citrate buffer, add neutral protease, and continue enzymatic hydrolysis at 50℃ and pH 6.8 for 2 hours. Then add flavor protease and continue enzymatic hydrolysis at 50℃ and pH 6.5 for 2 hours. After enzymatic hydrolysis, raise the temperature to 85℃ and maintain the temperature for 10 minutes to inactivate the enzyme. The amount of neutral protease added is 1.5 wt% of the pretreated yam protein powder, and the amount of flavor protease added is 1.0 wt% of the pretreated yam protein powder.

[0057] 3. After inactivation, two-stage coarse separation is performed: First, preliminary filtration is completed through a 200-mesh sieve, and then microfiltration is performed using a 0.2μm ceramic membrane to remove macromolecular impurities and insoluble residues; after coarse separation, fine separation is performed: at 0.2MPa, 25℃, and a flow rate of 2m / s, the solution is first filtered through a 10kDa ultrafiltration membrane, the permeate is collected, and unhydrolyzed macromolecular proteins and peptides above 10kDa in the system are removed. Then, the obtained permeate is filtered through a 1kDa ultrafiltration membrane, the retentate is collected, and fine separation is completed.

[0058] 4. After fine separation, the retentate is pre-concentrated using a reverse osmosis membrane. After concentration to 1 / 4 of the retentate volume, vacuum membrane concentration is carried out at 55℃ and 0.05MPa. After concentration three times, peptide powder is prepared by spray drying. The inlet air temperature is set to 180℃ and the outlet air temperature is set to 85℃. After spray drying, yam protein peptide powder is obtained.

[0059] 5. Add yam protein peptide powder to phosphate buffer solution with pH 9 at a solid-liquid ratio of 1g:100mL, stir at 400rpm for 30min, add gallic acid diluted with anhydrous ethanol under light-protected conditions, stir at 500rpm for 14h at 30℃, after the reaction is complete, centrifuge at 8000rpm for 10min at 4℃, and take the supernatant; the amount of gallic acid added is 7.5wt% of yam protein peptide powder; the concentration of gallic acid after dilution with anhydrous ethanol is 50mg / mL.

[0060] 6. Using a dialysis bag with a molecular weight cutoff of 1 kDa, dialyze the supernatant obtained in step 5 at 4°C for 24 hours, changing the ultrapure water every 6 hours. Collect the retentate and freeze-dry it at -30°C to obtain a food protein peptide with hypoglycemic activity.

[0061] 1. α-Amylase inhibition rate Prepare the following solutions: 0.02 mol / L, pH 6.0 phosphate buffer; prepare a 3 U / mL α-amylase working solution using the prepared phosphate buffer; substrate solution: prepare a 1% soluble starch solution using the prepared phosphate buffer; 2,3-dinitrosalicylic acid (DNS) reagent; food protein peptide solution: prepare a 0.5 mg / mL food protein peptide solution using the prepared phosphate buffer.

[0062] Set them separately: (1) Blank group: 0.4 mL phosphate buffer + 0.4 mL food protein peptide solution + 1.2 mL substrate solution + 0.5 mL DNS; (2) Control group: 0.4 mL phosphate buffer + 0.4 mL α-amylase working solution + 1.2 mL substrate solution, incubate at 37°C for 15 min, then add 0.5 mL DNS; (3) Experimental group: 0.4 mL of food protein peptide solution + 0.4 mL of α-amylase working solution, pre-incubate at 37℃ for 5 min, add 1.2 mL of substrate solution, incubate at 37℃ for 15 min, add 0.5 mL of DNS; The food protein peptide solution of the experimental group was replaced with an equal volume of 0.5 mg / mL acarbose as a positive control group. All groups were boiled in a water bath for 10 min and cooled to equilibrate for 10 min. The absorbance was measured at a wavelength of 540 nm. The inhibition rate of food protein peptides against α-amylase obtained from different examples and comparative examples was calculated. The test results are shown in Table 2.

[0063] Table 2. Inhibition rate of food protein peptides on α-amylase

[0064] As shown in Table 2, the hypoglycemic activity of the food protein peptides obtained in Examples 1-3 is superior to that in Comparative Examples 1-2. Comparative Example 1 lacks the synergistic effect of gallic acid and yam protein peptide, thus failing to competitively bind to the α-amylase active site through gallic acid, affecting the hydrolytic ability of α-amylase on starch and resulting in a decrease in the inhibition rate of α-amylase. Comparative Example 2 lacks chitosan-STPP encapsulation of gallic acid-modified yam protein peptide. The encapsulation structure can improve the stability of the protein peptide and reduce its conformational changes. Without the encapsulation, the conformation of the protein peptide is easily altered, reducing the hypoglycemic activity of the food protein peptide and causing a decrease in the inhibition rate of α-amylase.

[0065] 2. Inhibition rate of α-glucosidase Prepare the following solutions separately: 0.1 mol / L sodium carbonate solution; 0.05 mol / L, pH 6.8 tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer; enzyme solution: prepare 0.2 U / mL α-glucosidase solution using Tris-HCl buffer; substrate solution: prepare 5 mmol / L 4-nitrophenyl-β-D-glucopyranose (PNPG) solution using Tris-HCl buffer; food protein peptide solution: prepare 0.5 mg / mL food protein peptide solution using Tris-HCl buffer.

[0066] Set them separately: (1) Blank group: 50 μL Tris-HCl buffer + 50 μL food protein peptide solution + 50 μL PNPG solution + 50 μL sodium carbonate solution; (2) Control group: 50 μL Tris-HCl buffer + 50 μL enzyme solution, incubated at 37℃ for 10 min, 50 μL PNPG solution was added, incubated at 37℃ for 30 min, and 50 μL sodium carbonate solution was added; (3) Sample group: 50 μL of food protein peptide solution + 50 μL of enzyme solution, incubate at 37℃ for 10 min, add 50 μL of PNPG solution, incubate at 37℃ for 30 min, add 50 μL of sodium carbonate solution; The food protein peptide solution of the sample group was replaced with an equal volume of 0.5 mg / mL acarbose as a positive control group. The absorbance of each group was measured at a wavelength of 405 nm using an ELISA reader. The inhibition rate of the food protein peptides obtained in different examples and comparative examples against α-glucosidase was calculated. The test results are shown in Table 3.

[0067] Table 3. Inhibition rate of food protein peptides on α-glucosidase

[0068] As shown in Table 3, the food protein peptides obtained in Examples 1-3 showed higher inhibition rates against α-glucosidase than those in Comparative Examples 1-2. Comparative Example 1 lacked gallic acid modification of the yam protein peptide, exposing its active amino acid sites, making it prone to conformational aggregation and unable to effectively bind to the active site of α-glucosidase, thus reducing the inhibition rate. Comparative Example 2 lacked chitosan-STPP encapsulation of the food protein peptide, making it prone to conformational instability in the in vitro system, affecting its hypoglycemic activity and decreasing its inhibition rate against α-glucosidase.

[0069] 3. Free radical scavenging efficiency The food protein peptides obtained in each example and comparative example were prepared into a 0.5 mg / mL food protein peptide solution using phosphate buffer. 2.0 mL of the food protein peptide solution was transferred to a stoppered test tube, and 2.0 mL of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) solution (0.2 mmol / L) was added. The mixture was shaken well and reacted in the dark for 30 min. Then, 2.0 mL of the food protein peptide solution and 2.0 mL of anhydrous ethanol were mixed and reacted in the dark for 30 min. Finally, 2.0 mL of anhydrous ethanol and 2.0 mL of DPPH solution were mixed and reacted in the dark for 30 min. After the reaction was complete, the absorbance of each group was measured at 517 nm. The DPPH free radical scavenging rate of the food protein peptides obtained in different examples and comparative examples was calculated. The test results are shown in Table 4.

[0070] Table 4. Scavenging rate of DPPH free radicals by food protein peptides

[0071] Table 4 shows that the DPPH free radical scavenging rates of the food protein peptides obtained in Examples 1-3 were higher than those in Comparative Examples 1-2. After modification of the yam protein peptides with gallic acid, the phenolic hydroxyl groups in the gallic acid exhibit antioxidant activity. Simultaneously, the synergistic effect of gallic acid and yam protein peptides enhances their free radical scavenging ability, thereby improving the antioxidant capacity of the food protein peptides. Comparative Example 1 lacked gallic acid modification of the yam protein peptides, relying solely on the antioxidant activity of the yam protein peptides themselves, thus resulting in a decreased DPPH free radical scavenging rate. Comparative Example 2 lacked chitosan-STPP encapsulation of the food protein peptides; in vitro encapsulation has a relatively small impact on free radical scavenging ability, therefore the decrease in DPPH free radical scavenging rate was relatively small.

[0072] 4. Duration of drug action A type 2 diabetes mellitus (T2DM) mouse model was established by intraperitoneal injection of streptozotocin (STZ). Mice with successfully induced T2DM were randomly divided into six groups, with five groups receiving 20 mg / kg of streptozotocin. -1 ·d -1 Mice were fed food protein peptides, while the remaining group was given an equal amount of physiological saline as a model control group. The effects of food protein peptides obtained from different embodiments and comparative examples on fasting blood glucose (FBG) in mice were studied, and the test results are shown in Table 5.

[0073] Table 5 Results of FBG test in mice

[0074] As shown in Table 5, the hypoglycemic activity of the food protein peptides obtained in Examples 1-3 lasted longer. Although the fasting blood glucose of the diabetic model mice in Examples 1-3 did not decrease to normal levels after 35 days, it still showed a continuous downward trend. After the food protein peptides entered the alkaline environment of the intestine, the protonation degree of chitosan decreased under alkaline conditions, and the electrostatic cross-linking between chitosan and STPP weakened, causing the chitosan-STPP carrier structure to swell and loosen. The gallic acid-modified yam protein peptides embedded in it were slowly released, prolonging the hypoglycemic period. In Comparative Example 2, chitosan-STPP was not used as a nanocarrier. The gallic acid-modified yam protein peptides were released instantaneously. In the early stage, the mice had a greater decrease in FBG and lower blood glucose levels, but the hypoglycemic period was shorter. In the later stage, the FBG of the mice began to gradually rise.

[0075] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing food protein peptides with hypoglycemic activity, characterized in that, Includes the following steps: S1. Take solution A and solution B at a volume ratio of 5~7:

1. Add solution B dropwise to solution A at a rate of 0.5~1.0 mL / min with stirring. After the addition is complete, continue stirring for 2 hours. Then centrifuge at 4℃ and 8000 rpm. After centrifugation, discard the supernatant. Wash the precipitate with deionized water and centrifuge to obtain the solid phase. Resuspend the obtained solid phase with deionized water at a solid-liquid ratio of 1 g:10 mL to obtain a suspension. S2. Add mannitol to the suspension, mix thoroughly, pre-freeze at -80℃, and then freeze-dry at -50℃ and 10kPa to obtain food protein peptides with hypoglycemic activity; the amount of mannitol added is 5-8 wt% of the solid phase contained in the suspension. The A solution is composed of 1.5-2.0% acetic acid solution, chitosan, EDTA-2Na, and gallic acid-modified yam protein peptide powder. Solution B is a STPP solution with a mass concentration of 0.1~0.3%.

2. The method for preparing a food protein peptide with hypoglycemic activity according to claim 1, characterized in that, The preparation method of solution A is as follows: using 1.5~2.0% acetic acid solution as solvent, chitosan is added under stirring to prepare a chitosan solution with a concentration of 1.0~2.0 mg / mL. EDTA-2Na is added under stirring and completely dissolved. Gallic acid modified yam protein peptide powder is added and the pH is adjusted to 4.4~4.

6. After filtration through a filter membrane, solution A is obtained and placed at room temperature for later use.

3. The method for preparing a food protein peptide with hypoglycemic activity according to claim 2, characterized in that, The preparation method of the gallic acid modified yam protein peptide powder is as follows: S1. Under water bath heating at 45~50℃, add the pretreated yam protein powder to deionized water at a mass ratio of 1:15, adjust the pH to 6.5~6.9, and after enzymatic hydrolysis by neutral protease and flavor protease, raise the temperature to 85℃ and keep it constant for 10~15 minutes to inactivate the enzymes. S2. After inactivation, the product undergoes two-stage coarse separation followed by fine separation, then pre-concentration via reverse osmosis membrane, and finally vacuum membrane concentration at 55-60℃ for 3-5 times. After concentration, the product is spray-dried to prepare peptide powder, yielding yam protein peptide powder. S3. Add yam protein peptide powder to phosphate buffer solution with pH 9 at a solid-liquid ratio of 1g:100mL. Stir until completely dissolved, and then add gallic acid diluted with anhydrous ethanol under light-protected conditions. Stir and react at 30℃ for 14-16h. After the reaction is complete, centrifuge and take the supernatant. After dialyzing and freeze-drying, the obtained supernatant is used to obtain gallic acid modified yam protein peptide powder.

4. The method for preparing a food protein peptide with hypoglycemic activity according to claim 3, characterized in that, The pretreatment method for the yam protein powder is as follows: dissolve the yam protein powder in deionized water at a mass ratio of 1:10, adjust the pH to 6.5~7.0, add α-amylase, react at 60℃ for 30 min, and after the reaction is completed, centrifuge, wash, and vacuum dry to obtain the pretreated yam protein powder; the amount of α-amylase added is 0.3~0.5 wt% of the yam protein powder.

5. The method for preparing a food protein peptide with hypoglycemic activity according to claim 3, characterized in that, In step S1, the amount of neutral protease added is 1.5~2.0 wt% of the pretreated yam protein powder; the amount of flavor protease added is 1.0~1.5 wt% of the pretreated yam protein powder.

6. The method for preparing a food protein peptide with hypoglycemic activity according to claim 3, characterized in that, In step S2, the two-stage coarse separation method is as follows: first, preliminary filtration is completed through a 200-mesh sieve, and then microfiltration is performed using a 0.2μm ceramic membrane; the fine separation method is as follows: at 0.2MPa, 25℃, and a flow rate of 2~3m / s, the liquid is first filtered through a 10kDa ultrafiltration membrane, the permeate is collected, and then the obtained permeate is filtered through a 1kDa ultrafiltration membrane, the retentate is collected, and the fine separation is completed.

7. The method for preparing a food protein peptide with hypoglycemic activity according to claim 3, characterized in that, In step S3, the amount of gallic acid added is 7.5~12.5wt% of the yam protein peptide powder; the concentration of gallic acid after dilution with anhydrous ethanol is 50~60mg / mL.

8. The method for preparing a food protein peptide with hypoglycemic activity according to claim 2, characterized in that, The amount of gallic acid-modified yam protein peptide powder added is 10-30 wt% of chitosan; the mass ratio of chitosan to EDTA-2Na is 1:

1.

9. The method for preparing a food protein peptide with hypoglycemic activity according to claim 1, characterized in that, The preparation method of solution B is as follows: weigh STPP, dissolve it in deionized water and stir evenly to prepare a STPP solution with a mass concentration of 0.1~0.3%, filter it through a 0.22μm filter membrane to obtain solution B, and seal and store it for later use.

10. Food protein peptides with hypoglycemic activity prepared by the preparation method according to any one of claims 1 to 9.

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