A method for preparing millet polypeptide solution through cascade enzymolysis

Abstract

The present application relates to the technical field of food processing, and particularly relates to a method for preparing millet polypeptide liquid through gradient enzymolysis. The present application establishes a starch amylase liquidation saccharification-protease gradient combination enzymolysis process in view of the technical bottleneck that the starch colloid network after millet gelatinization hinders protease hydrolysis; through optimizing the protease gradient enzymolysis sequence, a specific scheme of first neutral protease and then alkaline protease is determined, the obtained millet polypeptide content is 1.02 mg / mL, the DPPH free radical scavenging rate reaches 89.63%, the reducing power is 0.26, and the inhibition rates of alpha-glucosidase and alpha-amylase in the concentration range of 0.2 mg / mL to 1.0 mg / mL are 62.3% to 81.9% and 52.0% to 80.0%, respectively. The present application effectively solves the common technical obstacles in the preparation of high-starch cereal polypeptides, and has excellent antioxidant activity and in-vitro hypoglycemic activity.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a method for preparing millet polypeptide liquid through stepwise enzymatic hydrolysis. Background Technology

[0002] Millet is one of the oldest cultivated crops, originating in the Yellow River basin with a cultivation history of over ten thousand years, holding an irreplaceable position in agricultural civilization. Millet possesses both nutritional value and health benefits, and is recorded in ancient medical texts such as the *Compendium of Materia Medica* as "nourishing stomach qi and replenishing deficiencies." Modern nutritional research shows that millet contains 9% to 14% protein, and its amino acid composition is superior to that of wheat and rice. It is rich in dietary fiber, B vitamins, and various minerals and micronutrients such as iron, zinc, and selenium, making it an ideal source of dietary nutrition.

[0003] Among the many functional active components of millet, bioactive peptides released from protein through enzymatic decomposition are particularly noteworthy. Bioactive peptides are short peptide fragments composed of 2-20 amino acid residues. Compared to original proteins and free amino acids, they have smaller molecular weights, higher transmembrane transport efficiency, and faster in vivo absorption. They often exert significant physiological regulatory functions even at very low doses, thus being considered excellent carriers of food functional factors. Numerous studies have confirmed that plant-derived bioactive peptides show broad application prospects in areas such as antioxidation, blood pressure reduction, immune regulation, antibacterial activity, blood sugar reduction, and improvement of lipid metabolism. Compared to soybean peptides and corn peptides, which have already achieved industrial-scale development, research on millet peptides is still in its early stages. Furthermore, the low allergenicity of millet protein gives it a natural advantage in special medical purpose formula foods and infant formula foods—characteristics that are difficult for other common grain peptides to possess simultaneously.

[0004] Despite the promising functional potential of millet peptides, current preparation technology bottlenecks severely restrict in-depth research on their activity and application development. In existing technologies, the preparation of millet peptide solutions typically involves gelatinizing millet flour with water and then directly adding protease for a one-step hydrolysis. However, in this process, the starch content of millet, which is over 70%, forms a dense colloidal network during moist heat treatment (such as gelatinization). This physical barrier significantly reduces the accessibility of the protein substrate to the protease, severely limiting the hydrolysis efficiency of the enzymatic hydrolysis system, resulting in low peptide content and insufficient functional activity in the obtained millet peptide solution. This problem is a common challenge faced by high-starch grains in peptide solution preparation—without breaking down the starch colloidal barrier, the protease cannot effectively contact and hydrolyze the encapsulated protein substrate.

[0005] Therefore, there is an urgent need to provide a method for preparing millet polypeptide liquid that can effectively break through the gelatinized starch colloidal barrier and directionally release highly active functional peptides. Summary of the Invention

[0006] To overcome the technical shortcomings of existing methods for preparing millet polypeptides, such as the gelatinized starch colloidal network hindering the efficiency of protease hydrolysis, this invention provides a method for preparing millet polypeptide liquid through stepwise enzymatic hydrolysis.

[0007] This invention is achieved through the following technical solution: A method for preparing millet polypeptide solution by stepwise enzymatic hydrolysis includes the following steps: The gelatinized system is heated to 90℃~100℃, and α-amylase and calcium chloride are added to obtain the liquefied system; the amount of α-amylase added is 0.2kg~0.4kg / ton of millet flour, and the amount of calcium chloride added is 1.0kg / ton~2.0kg / ton of millet flour.

[0008] The temperature of the liquefied system was lowered to 55℃~65℃, the pH was adjusted to 4.5~5.5, and β-amylase was added to obtain the saccharified system; the amount of β-amylase added was 0.5kg / ton~1.5kg / ton of millet flour.

[0009] Adjust the pH of the saccharified system to 6.5-7.5, add neutral protease, and enzymatically hydrolyze at 45℃-55℃ for 1.5-2.5 hours; then adjust the pH to 7.5-8.5, add alkaline protease, and continue enzymatic hydrolysis at 45℃-55℃ for 1.5-2.5 hours; the amount of neutral protease added is 2%-9% of the mass of millet flour; the amount of alkaline protease added is 2%-9% of the mass of millet flour.

[0010] After enzymatic hydrolysis, the enzyme is inactivated, and the supernatant is collected by centrifugation to obtain millet polypeptide solution.

[0011] Preferably, the system of millet flour gelatinized is prepared by the following method: Millet is crushed and passed through a 60-mesh sieve to obtain millet powder.

[0012] Add millet flour to water to obtain millet slurry; the ratio of millet flour to water is 1:17~22.

[0013] Heat the millet slurry to 85℃~95℃ and keep it heated for 20min~40min to obtain the system after millet flour gelatinization.

[0014] Preferably, the pH adjustment is performed using citric acid or sodium carbonate.

[0015] Preferably, the enzyme inactivation conditions are heating at 85℃~95℃ for 5min~15min.

[0016] Preferably, the centrifugation conditions are 3000 r / min to 5000 r / min for 10 min to 20 min.

[0017] Preferably, the α-amylase has an activity of 72,000 u / mL; the β-amylase has an activity of 700,000 u / mL; the neutral protease has an activity of 300,000 u / g; and the alkaline protease has an activity of 200,000 u / g.

[0018] The millet polypeptide liquid was prepared by the method described above.

[0019] The application of the millet polypeptide liquid in the preparation of millet polypeptide beverages that help with anti-oxidation and / or help maintain healthy blood sugar levels.

[0020] Preferably, the specific preparation method of the millet polypeptide beverage is as follows: The millet polypeptide solution was mixed with water at a volume ratio of 1:0.5~2 to obtain a mixture.

[0021] Add high-fructose corn syrup to the mixture at a rate of 0.15 g / 1 kg of mixture, and stir for 20 to 40 minutes to obtain millet polypeptide beverage.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention employs a “amylase liquefaction and saccharification-protease stepwise enzymatic hydrolysis” process. Through α-amylase and β-amylase, the high-viscosity starch macromolecules formed after gelatinization are degraded into soluble sugars in an orderly manner. This effectively breaks the restriction of the gelatinized starch colloidal network on the protein substrate, significantly improves the contact efficiency and hydrolysis efficiency between the protease and the substrate, and solves the common technical bottleneck in the preparation of peptides from high-starch grains.

[0023] 2. This invention systematically compares the effects of different protease sequence combinations on peptide yield and functional activity. It was found that while the specific sequence of neutral protease followed by alkaline protease resulted in a lower peptide yield (1.02 mg / mL) compared to the reverse sequence (1.23 mg / mL), its DPPH radical scavenging rate (89.63%) and reducing power (0.26) were significantly superior. Alkaline protease is a non-specific endonuclease, and preferential action can easily lead to over-cleavage of the active sequence. Neutral protease, on the other hand, has a relatively specific cleavage site, and preferential hydrolysis can moderately "cut" the protein molecule, first releasing specific active fragments. Subsequently, alkaline protease performs complementary cleavage on this basis, avoiding excessive degradation of the core active region and more conducive to generating stable peptides with hydrogen-donating and free radical-scavenging capabilities.

[0024] 3. The millet polypeptide solution prepared by this invention showed an inhibition rate of 62.3% to 81.9% against α-glucosidase and an inhibition rate of 52.0% to 80.0% against α-amylase in the concentration range of 0.2 mg / mL to 1.0 mg / mL, exhibiting a significant concentration-dependent effect and demonstrating good in vitro hypoglycemic activity.

[0025] 4. The process of this invention is simple, the conditions are mild, and it does not involve organic reagents, making it suitable for industrial production. It provides a referable technical solution for the high-value-added processing of millet resources and the green preparation of starchy grain active peptide beverages. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the millet polypeptide functional beverage of the present invention; Figure 2 This is a flowchart illustrating the preparation process of the millet polypeptide functional beverage of the present invention. Figure 3 This is a graph showing the effect of different enzyme addition amounts on the hydrolysis efficiency of neutral protease in this invention. Figure 4 This is a graph showing the effect of different substrate concentrations on the hydrolysis efficiency of neutral protease in this invention. Figure 5 This is a graph showing the effect of different pH values ​​on the hydrolysis efficiency of neutral protease according to the present invention. Figure 6 This is a graph showing the effect of different enzyme addition amounts on the hydrolysis effect of alkaline protease in this invention. Figure 7 This is a graph showing the effect of different substrate concentrations on the hydrolysis efficiency of alkaline protease in this invention. Figure 8 This is a graph showing the effect of different pH values ​​on the hydrolysis efficiency of alkaline protease according to the present invention. Figure 9 This is a curve showing the inhibition rate of the millet polypeptide against α-glucosidase in this invention. Figure 10 This is a curve showing the inhibition rate of the millet polypeptide against α-amylase in this invention. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0031] α-Amylase was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. β-amylase was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. Neutral protease was purchased from Nanning Pangbo Biotechnology Co., Ltd. The alkaline protease was purchased from Shandong Longkete Enzyme Preparation Co., Ltd. All other reagents or raw materials used in this invention are commercially available products.

[0032] Example 1 This embodiment provides a method for preparing millet polypeptide liquid and millet polypeptide beverage through stepwise enzymatic hydrolysis, including the following steps: S1. Take millet raw material, remove visible impurities and broken particles, wash thoroughly with clean water and drain, then grind it using a grinder and pass it through a 60-mesh sieve to obtain millet flour.

[0033] S2. Add the millet powder obtained in step S1 to water at a powder-to-water ratio of 1:20, stir and disperse for 45 minutes to allow the powder to fully absorb water and obtain millet slurry.

[0034] S3. Heat the millet slurry obtained in step S2 to 90°C and keep it heated for 30 minutes to obtain the gelatinized system.

[0035] S4. Raise the temperature of the gelatinized system from step S3 to 95℃, then add α-amylase (enzyme activity 72000u / mL) and calcium chloride. The amount of α-amylase added is 0.3kg / ton of millet flour; the amount of calcium chloride added is 1.5kg / ton of millet flour; under continuous stirring, enzymatic hydrolysis is carried out for 45 minutes to obtain the liquefied system.

[0036] S5. Reduce the temperature of the liquefied system from step S4 to 60℃, adjust the pH to 5 with 1 mol / L citric acid, and add β-amylase (enzyme activity of 700,000 u / mL) at a rate of 1 kg / ton of millet flour. Continue stirring and enzymatic hydrolysis for 4 hours to obtain the saccharified system.

[0037] S6. Stepwise enzymatic hydrolysis with proteases: First, adjust the pH of the saccharified system from step S5 to 7.0 using citric acid or sodium carbonate. Then, add neutral protease (300,000 u / g) at 3% of the millet flour mass and hydrolyze at 50℃ for 2 hours. Next, adjust the pH to 8. Then, add alkaline protease (200,000 u / g) at 3% of the millet flour mass and continue hydrolysis at 50℃ for another 2 hours.

[0038] S7. Enzyme inactivation and centrifugation: After enzymatic hydrolysis, heat at 90℃ for 10 min to inactivate the enzyme, centrifuge at 4000 r / min for 15 min, and take the supernatant to obtain millet polypeptide solution.

[0039] S8. Preparation, sterilization and filling: Mix the millet polypeptide liquid obtained in step S8 with purified water at a volume ratio of 1:1 to obtain a mixture; add fructose syrup at a ratio of 0.15g / 1kg of mixture, stir for 30min until completely dispersed; heat at 140℃ for 4s and then cool and fill to obtain millet polypeptide beverage.

[0040] Example 2 The pH was kept constant at 6, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of neutral protease added was set to 5%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0041] Example 3 The pH was kept constant at 6, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of neutral protease added was set to 7%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0042] Example 4 The pH was kept constant at 6, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of neutral protease added was set to 9%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0043] Example 5 The pH was kept constant at 8, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of alkaline protease added was set to 5%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0044] Example 6 The pH was kept constant at 8, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of alkaline protease added was set to 7%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0045] Example 7 The pH was kept constant at 8, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of alkaline protease added was set to 9%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0046] Example 8 Preparation of millet polypeptide solution: The ratio of millet powder to water is 1:17.

[0047] The millet slurry was heated to 85°C and kept at that temperature for 20 minutes to obtain the gelatinized system.

[0048] The gelatinized system was heated to 90°C, and α-amylase and calcium chloride were added to obtain the liquefied system.

[0049] The amount of α-amylase added is 0.2 kg / ton of millet flour, and the amount of calcium chloride added is 1.0 kg / ton of millet flour; the temperature of the liquefied system is lowered to 55°C, the pH is adjusted to 4.5, and β-amylase is added to obtain the saccharified system.

[0050] The amount of β-amylase added is 0.5 kg / ton of millet flour; the pH of the saccharified system is adjusted to 6.5, neutral protease is added, and enzymatic hydrolysis is carried out at 45°C for 1.5 h; then the pH is adjusted to 7.5, alkaline protease is added, and enzymatic hydrolysis is continued at 45°C for 1.5 h.

[0051] The remaining steps are exactly the same as in Example 1.

[0052] Preparation of millet polypeptide beverage: The obtained millet polypeptide solution was mixed with purified water at a volume ratio of 1:0.5.

[0053] The remaining steps are exactly the same as in Example 1.

[0054] Example 9 Preparation of millet polypeptide solution: The ratio of millet powder to water is 1:22.

[0055] The millet slurry was heated to 95°C and kept at that temperature for 40 minutes to obtain the gelatinized system.

[0056] The gelatinized system was heated to 100°C, and α-amylase and calcium chloride were added to obtain the liquefied system. The amount of α-amylase added was 0.4 kg / ton of millet flour, and the amount of calcium chloride added was 2.0 kg / ton of millet flour.

[0057] The temperature of the liquefied system was lowered to 65℃, the pH was adjusted to 5.5, and β-amylase was added to obtain the saccharified system; the amount of β-amylase added was 1.5 kg / ton of millet flour.

[0058] Adjust the pH of the saccharified system to 7.5, add neutral protease, and enzymatically hydrolyze at 55°C for 2.5 h; then adjust the pH to 8.5, add alkaline protease, and continue enzymatic hydrolysis at 55°C for 2.5 h.

[0059] The remaining steps are exactly the same as in Example 1.

[0060] Preparation of millet polypeptide beverage: The obtained millet polypeptide solution was mixed with purified water at a volume ratio of 1:2.

[0061] The remaining steps are exactly the same as in Example 1.

[0062] Comparative Example 1 The pH was kept constant at 6, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of neutral protease added was set to 0.5%, where the substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0063] Comparative Example 2 The pH was kept constant at 6, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of neutral protease added was set to 1%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0064] The millet polypeptide solutions obtained in Examples 1-4 and Comparative Examples 1-2 were compared experimentally, and the results are as follows: Figure 3 As shown, the concentration of millet peptides increases with the increase of enzyme addition, and the peptide concentration tends to stabilize when the enzyme addition reaches 3%. Therefore, the addition amount of neutral protease is 3%~9%, and the optimal enzyme addition amount is 3%.

[0065] Comparative Example 3 The pH was kept constant at 6, the temperature at 50°C, the amount of neutral protease added at 3%, and the reaction time at 2 hours. The substrate concentration was set to 4%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0066] Comparative Example 4 The pH was kept constant at 6, the temperature at 50°C, the amount of neutral protease added at 3%, and the reaction time at 2 hours. The substrate concentration was set to 6%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0067] Comparative Example 5 The pH was kept constant at 6, the temperature at 50°C, the amount of neutral protease added at 3%, and the reaction time at 2 hours. The substrate concentration was set to 7%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0068] Comparative Example 6 The pH was kept constant at 6, the temperature at 50°C, the amount of neutral protease added at 3%, and the reaction time at 2 hours. The substrate concentration was set to 8%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0069] The millet polypeptide solutions obtained in Example 1 and Comparative Examples 3-6 were compared experimentally, and the results are as follows: Figure 4 As shown, the concentration of millet peptides first increases and then decreases with increasing substrate concentration, reaching a maximum of 0.54 mg / mL at a substrate concentration of 5%. Therefore, the optimal substrate concentration is 5%.

[0070] Comparative Example 7 The neutral protease addition was fixed at 3%, the temperature at 50°C, the substrate concentration at 5%, and the reaction time at 2 hours. The pH was set to 6. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0071] Comparative Example 8 The neutral protease addition was fixed at 3%, the temperature at 50°C, the substrate concentration at 5%, and the reaction time at 2 hours. The pH was set to 8. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0072] Comparative Example 9 The neutral protease addition was fixed at 3%, the temperature at 50°C, the substrate concentration at 5%, and the reaction time at 2 hours. The pH was set to 9. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0073] Comparative Example 10 The neutral protease addition was fixed at 3%, the temperature at 50°C, the substrate concentration at 5%, and the reaction time at 2 hours. The pH was set to 10. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0074] The millet polypeptide solutions obtained in Example 1 and Comparative Examples 7-10 were compared experimentally, and the results are as follows: Figure 5 As shown, the peptide concentration initially increases and then decreases with increasing pH, reaching a maximum of 0.57 mg / mL at pH 7. Therefore, the optimal pH for neutral proteases is 7.

[0075] Comparative Example 11 The pH was kept constant at 8, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of alkaline protease added was set to 0.5%, where the substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0076] Comparative Example 12 The pH was kept constant at 8, the temperature at 50°C, the substrate concentration at 5% (w / v), and the reaction time at 2 hours. The amount of alkaline protease added was set to 1%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0077] The millet polypeptide solutions obtained in Examples 1, 5-7, and Comparative Examples 11-12 were compared, and the results are as follows: Figure 6 As shown, when the enzyme addition is less than 3%, the peptide concentration increases with increasing enzyme addition; when the enzyme addition is greater than 3%, the peptide concentration hardly increases anymore. Therefore, the optimal addition amount of alkaline protease is 3%–9%, ​​with 3% being the most suitable.

[0078] Comparative Example 13 The pH was kept constant at 8, the temperature at 50°C, the alkaline protease dosage at 3%, and the reaction time at 2 hours. The substrate concentration was set to 4%. Substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0079] Comparative Example 14 The pH was kept constant at 8, the temperature at 50°C, the amount of alkaline protease added at 3%, and the reaction time at 2 hours. The substrate concentration was set to 6%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0080] Comparative Example 15 The pH was kept constant at 8, the temperature at 50°C, the amount of alkaline protease added at 3%, and the reaction time at 2 hours. The substrate concentration was set to 7%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0081] Comparative Example 16 The pH was kept constant at 8, the temperature at 50°C, the amount of alkaline protease added at 3%, and the reaction time at 2 hours. The substrate concentration was set to 8%. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0082] The millet polypeptide solutions obtained in Example 1 and Comparative Examples 13-16 were compared experimentally, and the results are as follows: Figure 7 As shown, the concentration of millet peptides first increases and then decreases with increasing substrate concentration, with the best hydrolysis effect observed at a substrate concentration of 5%. Therefore, the optimal substrate concentration is 5%.

[0083] Comparative Example 17 The alkaline protease addition was fixed at 3%, the temperature at 50°C, the substrate concentration at 5%, and the reaction time at 2 hours. The pH was set to 6. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0084] Comparative Example 18 The alkaline protease addition was fixed at 3%, the temperature at 50°C, the substrate concentration at 5%, and the reaction time at 2 hours. The pH was set to 7. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0085] Comparative Example 19 The alkaline protease addition was fixed at 3%, the temperature at 50°C, the substrate concentration at 5%, and the reaction time at 2 hours. The pH was set to 9. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0086] Comparative Example 20 The alkaline protease addition was fixed at 3%, the temperature at 50°C, the substrate concentration at 5%, and the reaction time at 2 hours. The pH was set to 10. The substrate concentration refers to the concentration of millet flour in water. The remaining steps were exactly the same as in Example 1.

[0087] The millet polypeptide solutions obtained in Example 1 and Comparative Examples 17-20 were compared experimentally, and the results are as follows: Figure 8 As shown, the peptide yield initially increases and then decreases with increasing pH, reaching its highest concentration at pH 8. Therefore, the optimal pH for alkaline protease is 8.

[0088] Comparative Example 21 Neutral protease was added and enzymatically hydrolyzed at pH 7 and 50°C for 2 hours; alkaline protease was not added, and the remaining steps were exactly the same as in Example 1.

[0089] Comparative Example 22 Alkaline protease was added and enzymatically hydrolyzed at pH 8 and 50°C for 2 hours; neutral protease was not added, and the remaining steps were exactly the same as in Example 1.

[0090] Comparative Example 23 First, add alkaline protease and hydrolyze at pH 8 and 50°C for 2 hours; then add neutral protease and hydrolyze at pH 7 and 50°C for 2 hours. The remaining steps are exactly the same as in Example 1.

[0091] The concentration of peptides obtained from the millet peptide solutions in Examples 1 and Comparative Examples 21-23 was determined using the biuret method. The DPPH free radical scavenging rate and reducing power of the peptides obtained from each method were also measured. The results are shown in Table 1.

[0092] Table 1. Peptide concentration and antioxidant capacity of different enzymatic hydrolysis processes Comparative Example 21 0.55 60.83 0.17 Comparative Example 22 0.59 58.43 0.18 Example 1 1.02 89.63 0.26 Comparative Example 23 1.23 68.98 0.21 As shown in Table 1, although the peptide yield of Example 1 (1.02 mg / mL) was lower than that of Comparative Example 23 (1.23 mg / mL), its DPPH free radical scavenging rate (89.63%) and reducing power (0.26) were significantly better than those of Comparative Example 23 and other comparative examples. This result indicates that although the stepwise combination sequence of neutral protease followed by alkaline protease does not offer an advantage in terms of the total peptide bond cleavage, its enzymatic pathway is more conducive to the targeted release of active peptides with hydrogen-donating and free radical-scavenging capabilities, avoiding excessive degradation of the core functional regions, thus exhibiting significant superiority in functional activity.

[0093] Example 10 Comparison of peptide content at different stages In this embodiment, the polypeptide content of millet polypeptide liquid after saccharification (before protease hydrolysis) and after enzymatic hydrolysis in Example 1 was measured to verify the rationality of the process design of the present invention.

[0094] To improve the peptide content obtained by this method, the present invention determined the peptide content before and after enzymatic hydrolysis, as shown in Table 2.

[0095] Table 2 Comparison of peptide content at different stages Peptide concentration (mg / mL) 0.70 1.02 The peptide content before enzymatic hydrolysis was low (0.70 mg / mL), ruling out interference from non-specific hydrolysis of the substrate protein by amylase, thus validating the safety and necessity of the initial amylase treatment. After enzymatic hydrolysis with a stepwise combination of proteases, the peptide content significantly increased to 1.02 mg / mL. These results indicate that the main function of the initial amylase liquefaction and saccharification treatment is to break the encapsulation restriction of the protein substrate by the gelatinized starch colloidal network, thereby improving the accessibility of the protease. Protease hydrolysis is the decisive step in the release of millet peptides.

[0096] Example 11 In vitro antioxidant activity assay of millet peptides This embodiment measures the in vitro antioxidant activity of the millet polypeptide liquid obtained in Example 1.

[0097] S1, Reducing power measurement Take 1 mL of a 1 mg / mL millet polypeptide solution, add 1 mL of 1% potassium ferricyanide and 1 mL of pH 6.6 phosphate buffer, mix well, and incubate at 50℃ for 20 min. Add 2.5 mL of 10% trichloroacetic acid solution and centrifuge at 3000 rpm for 10 min. Take 2.5 mL of the supernatant, add 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride, mix well, let stand for 10 min, and measure the absorbance at 700 nm. Higher absorbance indicates stronger reducing power. The results show that the reducing power of the millet polypeptide obtained in this invention is 0.26 at a concentration of 1.00 mg / mL.

[0098] S2 and DPPH free radical scavenging capacity determination Mix 2 mL of DPPH solution with 2 mL of sample solution thoroughly, and react at 37 °C in the dark for 30 min. Measure the absorbance A1 at 517 nm. Use anhydrous ethanol instead of DPPH solution and 50% ethanol instead of sample solution as blank controls, and measure the absorbance A2 and A3 respectively. Calculate the DPPH free radical scavenging rate according to the formula:

[0099] DPPH free radical scavenging rate (%) = (1-(A1-A2) / A3)×100%.

[0100] The results showed that the millet peptide obtained in this invention had a DPPH free radical scavenging rate of 89.63% at a concentration of 1.00 mg / mL.

[0101] Example 12 In vitro hypoglycemic activity assay of millet peptides This embodiment measures the in vitro hypoglycemic activity of the millet polypeptide solution obtained in Example 1. Its inhibition rates against α-glucosidase and α-amylase were measured respectively.

[0102] S1, α-glucosidase inhibition rate: A 5 mmol / L PNPG solution was prepared using neutral sodium phosphate buffer and mixed thoroughly with different concentrations of millet polypeptide sample solutions at a 2:1 ratio, denoted as B1. After incubating at 37℃ for 5 min, 10 μL of 1 U / mL α-glucosidase solution was added and mixed well. The mixture was then incubated in a water bath for 10 min. 140 μL of a 0.2 mmol / L Na2CO3 solution prepared using neutral sodium phosphate buffer was added to terminate the reaction, yielding the test sample solution. Using the buffer solution instead of the sample solution and the buffer solution instead of α-glucosidase, the above steps were repeated, denoted as B2 and B0 respectively. Acarbose was used as a positive control, and the absorbance of the above solutions was measured at a wavelength of 400 nm. The inhibition rate of millet polypeptide against α-glucosidase was calculated.

[0103] .

[0104] S2. Determination of α-amylase inhibition rate: Different concentrations of millet polypeptide sample solutions prepared with 20 mmol / L neutral sodium phosphate buffer were thoroughly mixed with 1 U / mL α-amylase at a 1:1 ratio, denoted as C1. After incubating in a 37℃ hot water bath for 10 min, 400 μL of 0.25% starch solution was added and mixed well. The mixture was then incubated in a water bath for 15 min. 1 mL of DNS colorimetric solution prepared with neutral sodium phosphate buffer was added to terminate the reaction. Finally, 10 mL of distilled water was added to obtain the test sample solution. Using the buffer solution instead of the sample solution and the buffer solution instead of α-amylase, the above steps were repeated, denoted as C2 and C0 respectively. Acarbose was used as a positive control. The absorbance of the above solutions was measured at a wavelength of 540 nm. The inhibition rate of millet polypeptide against α-amylase was calculated.

[0105] .

[0106] To improve the inhibitory performance obtained by this method, this invention systematically compared key parameters of the inhibition rates of millet peptides against α-glucosidase and α-amylase. For example... Figure 9 , Figure 10 As shown.

[0107] like Figure 9 , 10 As shown, within the concentration range of 0.2 mg / mL to 1.0 mg / mL, the millet peptide obtained in this invention exhibited an inhibition rate of 62.3% to 81.9% against α-glucosidase and 52.0% to 80.0% against α-amylase. The inhibition rate increased with increasing concentration, showing a clear concentration dependence. This result indicates that the millet peptide obtained in this invention possesses good in vitro hypoglycemic activity.

[0108] Example 13 Sensory quality of millet peptide beverage This embodiment evaluates the sensory quality of the millet polypeptide beverage prepared in Example 1.

[0109] Ten sensory evaluators were organized to assess the color, aroma, taste, and texture of the millet polypeptide beverage. A 10-point scale was used (1-3 points for poor, 4-6 points for average, 7-9 points for good, and 10 points for excellent). The results showed that the millet polypeptide beverage was a uniform light yellow liquid with the characteristic aroma of millet, a smooth taste, no unpleasant odors, a uniform and stable texture, and no sedimentation or layering. All sensory scores were above 8 points.

[0110] It should be noted that the results of in vitro antioxidant activity and in vitro hypoglycemic activity determination of the millet polypeptide liquid prepared in Examples 2-9 are the same as those of Example 1; the sensory quality evaluation of the millet polypeptide beverage prepared in Examples 2-9 is the same as that of Example 1, so there is no need to elaborate further.

[0111] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0112] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A method for preparing millet polypeptide liquid through stepwise enzymatic hydrolysis, characterized in that, Includes the following steps: The gelatinized millet flour system is heated to 90℃~100℃, and α-amylase and calcium chloride are added to obtain the liquefied system; the amount of α-amylase added is 0.2kg / ton~0.4kg / ton of millet flour, and the amount of calcium chloride added is 1.0kg / ton~2.0kg / ton of millet flour. The temperature of the liquefied system was lowered to 55℃~65℃, the pH was adjusted to 4.5~5.5, and β-amylase was added to obtain the saccharified system; the amount of β-amylase added was 0.5kg / ton~1.5kg / ton of millet flour; Adjust the pH of the saccharified system to 6.5-7.5, add neutral protease, and enzymatically hydrolyze at 45℃-55℃ for 1.5-2.5 hours; then adjust the pH to 7.5-8.5, add alkaline protease, and continue enzymatic hydrolysis at 45℃-55℃ for 1.5-2.5 hours; the amount of neutral protease added is 2%-9% of the mass of millet flour; the amount of alkaline protease added is 2%-9% of the mass of millet flour. After enzymatic hydrolysis, the enzyme is inactivated, and the supernatant is collected by centrifugation to obtain millet polypeptide solution.

2. The method according to claim 1, characterized in that, The system obtained after gelatinizing the millet flour was prepared by the following method: Millet is ground and passed through a 60-mesh sieve to obtain millet powder; Add millet flour to water to obtain millet slurry; the ratio of millet flour to water is 1:17~22. Heat the millet slurry to 85℃~95℃ and keep it heated for 20min~40min to obtain the system after millet flour gelatinization.

3. The method according to claim 1, characterized in that, The pH is adjusted using citric acid or soda ash.

4. The method according to claim 1, characterized in that, The enzyme inactivation conditions are heating at 85℃~95℃ for 5min~15min; the centrifugation conditions are centrifugation at 3000r / min~5000r / min for 10min~20min.

5. The method according to claim 1, characterized in that, The α-amylase has an activity of 72,000 u / mL; the β-amylase has an activity of 700,000 u / mL; the neutral protease has an activity of 300,000 u / g; and the alkaline protease has an activity of 200,000 u / g.

6. The millet polypeptide solution prepared by the method according to any one of claims 1 to 5.

7. The use of the millet polypeptide liquid according to claim 6 in the preparation of millet polypeptide beverages that help with antioxidation and / or help maintain healthy blood sugar levels.

8. The application according to claim 7, characterized in that, The specific preparation method of the millet polypeptide beverage is as follows: The millet polypeptide solution was mixed with water at a volume ratio of 1:0.5~2 to obtain a mixture. Add high-fructose corn syrup to the mixture at a rate of 0.15 g / kg of mixture, and stir for 20 to 40 minutes to obtain millet polypeptide beverage.

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