Preparation method of synergistic hypoglycemic tartary buckwheat flour rich in flavone and DPP-IV inhibitory peptide

Abstract

The invention discloses preparation of synergistic blood glucose reducing tartary buckwheat powder rich in flavone and DPP-IV inhibitory peptide and three tartary buckwheat protein source DPP-IV inhibitory peptide, and belongs to the technical field of food science. According to the invention, actual hydrolysis is guided by virtual hydrolysis, and multi-condition step-by-step screening is carried out by a computer, so that the novel tartary buckwheat polypeptide with obvious DPP-IV inhibitory activity is provided, and the peptide sequences are CPPL, IF and PIW. The germinated tartary buckwheat rich in flavonoid compounds is used as an enzymolysis substrate, tartary buckwheat germination and protease hydrolysis are combined for use, and the content of flavone with hypoglycemic activity and DPP-IV inhibitory peptide in tartary buckwheat powder is increased to the maximum extent. According to the invention, the problems of insufficient conversion of blood-sugar-reducing flavone components in the existing preparation of the tartary buckwheat powder and time-consuming and blind screening of traditional DPP-IV inhibitory active peptides are solved, and reliable material and theoretical basis is laid for development of tartary buckwheat blood-sugar-reducing functional products.

Description

Technical Field

[0001] The invention relates to the field of food science and technology, and in particular to the preparation of synergistic hypoglycemic tartary buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides and novel tartary buckwheat-derived DPP-IV inhibitory peptides. Background Art

[0002] The prevalence of diabetes has been rising steadily worldwide, and is expected to increase to 700 million in 2045, a 55% increase from 2019. It is considered one of the most serious chronic diseases. Among them, type 2 diabetes accounts for nearly 90%, and its onset is mainly related to insulin resistance and decreased secretory function of pancreatic β cells. DPP-IV inhibitors can promote insulin secretion by reducing the decomposition of GLP-1 and increasing the concentration of GLP-1. They are a new and effective treatment for type 2 diabetes. At present, a variety of food-derived small molecule peptides have been reported to have DPP-IV inhibitory activity, such as KL and LR from natto and IP and LP from rice. Its non-toxic side effects and excellent biocompatibility make it of great application value in the treatment of type 2 diabetes.

[0003] Tartary buckwheat is an internationally recognized grain that is both a medicine and food. It is rich in protein, dietary fiber and flavonoids and has the effect of regulating blood sugar. Tartary buckwheat protein contains 8 essential amino acids for the human body and is a rare high-quality plant protein that can improve the function of pancreatic β cells and reduce insulin resistance. However, the allergenic protein in tartary buckwheat can cause allergic reactions in the skin and digestive system. Tartary buckwheat protein peptides are obtained by protease digestion and are easier to absorb, safer and more biologically active than tartary buckwheat protein. Flavonoids, as secondary metabolites, are the main components of tartary buckwheat's hypoglycemic activity and are mainly synthesized through the phenylpropanoid metabolic pathway. Germination treatment can inhibit the activity of rutin degrading enzymes and increase the activity of the key enzyme for flavonoid synthesis (phenylalanine ammonia lyase, PAL), thereby effectively enriching tartary buckwheat flavonoids; in addition, germination treatment can also improve the digestion characteristics of tartary buckwheat and reduce or eliminate the content of anti-nutrients.

[0004] Traditional methods for screening and identifying active peptides include continuous fractionation, purification and mass spectrometry identification, which are complex and time-consuming. The development of computers and bioinformatics can provide a guiding reference for the rapid acquisition and screening of umami peptides.

[0005] Therefore, using sprouted buckwheat as raw material and guided by the results of virtual hydrolysis, a specific protease was used to develop a preparation method for synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides, so as to maximize the content of flavonoids with hypoglycemic activity and high-activity DPP-IV inhibitory peptides in buckwheat flour, in order to obtain buckwheat flour rich in flavonoids and high DPP-IV inhibitory active peptides, solve the problem that the hypoglycemic function of the existing preparation of buckwheat flour is not outstanding, and achieve a hypoglycemic effect of 1+1>2, which is a problem that technicians in this field urgently need to solve. Summary of the invention

[0006] To this end, the present invention provides a method for preparing synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides, so as to solve the problems in the prior art that the hypoglycemic function of the prepared buckwheat flour is not prominent enough and the screening and utilization of DPP-IV inhibitory peptide active peptides is inaccurate and slow.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] According to the present invention, a method for preparing a synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides comprises the following steps:

[0009] S1. Ultrasonic pretreatment: Wash and dry the bitter buckwheat, sterilize it and then perform ultrasonic treatment;

[0010] S2. Soaking the buckwheat: placing the tartary buckwheat after ultrasonic pretreatment at a constant temperature of 25±5℃ for two intermittent soaking treatments;

[0011] S3. Germination: The soaked bitter buckwheat was intermittently cultured under constant temperature and humidity conditions, sprayed with 3-6% sucrose solution every 2-4 hours, and germinated for 2-4 days to obtain germinated bitter buckwheat;

[0012] S4. Washing and beating: After washing the buckwheat buds with clean water, adding drinking water at a weight ratio of 1:10-1:15 for beating, and filtering to prepare a filtered slurry;

[0013] S5. Virtual hydrolysis: Collect the amino acid sequences of buckwheat proteins, use single or double enzymes to virtually hydrolyze the sequences through the database, and predict the DPP-IV inhibitory activity of the resulting peptides;

[0014] S6. In vitro hydrolysis: According to the guiding parameters obtained in S5, the filtrate obtained in S4 is heated, cooled to room temperature, the solution is adjusted to the optimal pH value of the protease, and the protease is added according to the enzyme-substrate ratio and then hydrolyzed;

[0015] S7. Sequence identification: After the enzymatic hydrolysate cooled to room temperature is centrifuged, the supernatant is freeze-dried for sequence identification;

[0016] S8. DPP-IV inhibitory peptide screening: Based on the identification results of S7, a series of screening conditions are used to screen the obtained peptides step by step to obtain specific peptides with high DPP-IV inhibitory activity;

[0017] S9. Drying: The enzymatically hydrolyzed liquid is concentrated and spray-dried, or dried using other techniques such as freeze-drying.

[0018] Furthermore, the specific steps of virtual hydrolysis in S5 are as follows: collecting the amino acid sequence of tartary buckwheat protein, using the BIOPEP-UWM database to virtually hydrolyze the sequence and predict the DPP-IV activity of the obtained peptide.

[0019] Furthermore, the specific steps of in vitro hydrolysis in S6 are as follows: heating the filtered slurry obtained in S4 at 90-100°C for 10-15 minutes, stirring continuously during the period to ensure that the protein is completely denatured by heat; after cooling to room temperature, using NaOH or HCl to adjust the solution to the optimal pH value of the protease; adding protease according to the enzyme-substrate ratio: 6000-10000U / g protein for hydrolysis for 4-8 hours; after the hydrolysis is completed, heating in a boiling water bath to terminate the hydrolysis reaction.

[0020] Furthermore, the specific steps of sequence identification in S7 are as follows:

[0021] Take 20-30 mL of the enzymatic hydrolysate cooled to room temperature, centrifuge at 6000-8000 rpm for 15-20 min, take the supernatant for freeze-drying, and use LC-MS / MS for sequence identification.

[0022] Furthermore, the screening conditions in S8 are specifically molecular docking, activity prediction, physicochemical property prediction, sensitization and toxicity prediction, and amino acid composition distribution.

[0023] Furthermore, the ultrasonic pretreatment in S1 comprises the following steps:

[0024] (1) Optimization and cleaning: Select high-quality bitter buckwheat produced in the current year, select those with full grains for later use, and clean and dry them;

[0025] (2) Disinfection: Soak the treated buckwheat in a 5% to 8% sodium hypochlorite solution for 10 to 15 minutes;

[0026] (3) Ultrasonic pretreatment: The sterilized buckwheat was placed under ultrasound with a power of 25 kHz for 12-16 min.

[0027] Furthermore, the specific process of the intermittent soaking treatment in S2 is: soaking the bitter buckwheat in a 3% sucrose solution for 2-4 hours and then taking it out for ventilation for 6-9 hours; the intermittent culture treatment process in S3 is: irradiating the bitter buckwheat after soaking for 12 hours and then treating it in the dark for 12 hours.

[0028] Furthermore, the protease in S5 is selected from one or more of alkaline protease, papain, neutral protease, bromelain and trypsin.

[0029] Furthermore, the hydrolysis conditions selected in S6 are performed and determined based on the virtual hydrolysis results of S5, and the method for determining the degree of hydrolysis is formaldehyde titration.

[0030] Furthermore, in the S9, the spray drying air inlet temperature is 170±10°C, the air outlet temperature is 80±5°C, the atomizer speed is 15000r / min, and the feed rate is 70±5mL / min.

[0031] The present invention has the following advantages:

[0032] 1. The buckwheat flour in this application is treated with germination and protease hydrolysis to achieve efficient conversion of flavonoids while maximizing the content of DPP-IV inhibitory peptides in the buckwheat flour, achieving a hypoglycemic effect of 1+1>2.

[0033] 2. This application is guided by computer virtual hydrolysis results, simplifies in vitro enzymatic hydrolysis conditions, and solves the problem of inaccurate and slow screening of DPP-IV inhibitory peptide active peptides in the prior art.

[0034] 3. This application comprehensively scores the peptides identified by mass spectrometry through molecular docking, activity, toxicity and physicochemical properties, and obtains three new peptides with the strongest DPP-IV inhibitory activity, namely CPPL, IF, and PIW, which can form hydrogen bonding forces with amino acid residues such as TYR666, TYR662, and GLU206 in the active pocket of the receptor protein. This is the key interaction force for the three peptides to have DPP-IV inhibitory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0036] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0037] Figure 1 is a preparation flow chart of the present invention;

[0038] Figure 2 A demonstration diagram of peptide docking of a protein after the solvent and organic matter are removed provided by the present invention;

[0039] Figure 3This is a diagram showing the results of protein docking provided by the present invention. DETAILED DESCRIPTION

[0040] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The present application provides a method for preparing synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides, comprising the following steps:

[0042] S1. Ultrasonic pretreatment: Wash and dry the bitter buckwheat, sterilize it and then perform ultrasonic treatment;

[0043] S2. Soaking the buckwheat: placing the tartary buckwheat after ultrasonic pretreatment at a constant temperature of 25±5℃ for two intermittent soaking treatments;

[0044] S3. Germination: The soaked bitter buckwheat was intermittently cultured under constant temperature and humidity conditions, sprayed with 3-6% sucrose solution every 2-4 hours, and germinated for 2-4 days to obtain germinated bitter buckwheat;

[0045] S4. Washing and beating: After washing the buckwheat buds with clean water, adding drinking water at a weight ratio of 1:10-1:15 for beating, and filtering to prepare a filtered slurry;

[0046] S5. Virtual hydrolysis: Collect the amino acid sequences of buckwheat proteins, perform virtual hydrolysis on the sequences through the database and predict the DPP-IV inhibitory activity of the resulting peptides;

[0047] S6. In vitro hydrolysis: According to the parameters and results obtained in S5, the filtrate obtained in S4 is heated, cooled to room temperature, the solution is adjusted to the optimal pH value of the protease, and then the protease is added according to the enzyme-substrate ratio and then hydrolyzed;

[0048] S7. Sequence identification: After the enzymatic hydrolysate cooled to room temperature is centrifuged, the supernatant is freeze-dried for sequence identification;

[0049] S8. Screening of DPP-IV inhibitory peptides: Based on the results identified in S7, a series of conditions including molecular docking, activity prediction, physicochemical property prediction, sensitization and toxicity prediction, and amino acid composition were used for step-by-step screening;

[0050] S9. Drying: The enzymatically hydrolyzed liquid is concentrated and spray-dried, or other drying techniques such as freeze-drying are used.

[0051] Specifically, the specific steps of virtual hydrolysis are as follows: collect the amino acid sequence of buckwheat protein, use the BIOPEP-UWM database to virtually hydrolyze the sequence and predict the DPP-IV inhibitory activity of the obtained peptide.

[0052] The specific steps of in vitro hydrolysis are as follows: heat the filtered slurry obtained from S4 at 90-100°C for 10-15 minutes, stirring continuously during the process to ensure that the protein is completely denatured by heat; after cooling to room temperature, use NaOH or HCl to adjust the solution to the optimal pH value for the protease; add protease at an enzyme-substrate ratio of 6000-10000U / g protein for hydrolysis for 4-8 hours; after the hydrolysis is completed, heat in a boiling water bath to terminate the hydrolysis reaction.

[0053] The specific steps of sequence identification are as follows:

[0054] Take 20-30 mL of the enzymatic hydrolysate cooled to room temperature, centrifuge at 6000-8000 rpm for 15-20 min, take the supernatant for freeze-drying, and use LC-MS / MS for sequence identification.

[0055] Ultrasonic pretreatment includes the following steps:

[0056] (1) Selection and cleaning: Select high-quality buckwheat produced that year, pick out those with full grains for later use, and then clean and dry them.

[0057] (2) Disinfection: Soak the treated bitter buckwheat in a 5%-8% sodium hypochlorite solution for 10-15 minutes.

[0058] (3) Ultrasonic pretreatment: The sterilized buckwheat was placed under ultrasound with a power of 25 kHz for 12-16 minutes;

[0059] Specifically, the specific process of the intermittent soaking treatment in S2 is: soaking the bitter buckwheat in a 3% sucrose solution for 2-4 hours and then taking it out for ventilation for 6-9 hours;

[0060] The intermittent culture process in S3 is as follows: the soaked buckwheat is exposed to light for 12 hours and then kept in darkness for 12 hours.

[0061] Among them, the protease in S5 is selected from one or more of alkaline protease, papain, neutral protease, trypsin and bromelain.

[0062] Among them, the method for determining the hydrolysis degree in S6 is the formaldehyde titration method.

[0063] In S9, the spray drying inlet temperature is 170±10°C, the outlet temperature is 80±5°C, the atomizer speed is 15000r / min, and the feed rate is 70±5mL / min. As an optional method, other drying methods such as freeze drying can also be used in this application.

[0064] Example 1: Preparation method of flavonoid-rich budding buckwheat flour

[0065] S1. Ultrasonic pretreatment: Select high-quality bitter buckwheat produced in the current year, select bitter buckwheat with full grains, and wash and dry them;

[0066] Soak the treated bitter buckwheat in 8% sodium hypochlorite solution for 15 minutes;

[0067] The sterilized bitter buckwheat was placed under ultrasound with a power of 25 kHz for 16 minutes.

[0068] S2. Soaking: The bitter buckwheat after ultrasonic pretreatment is placed under a constant temperature of 25° C. for two intermittent soaking treatments. The specific process of the intermittent soaking treatment is: soak the bitter buckwheat in a 3% sucrose solution for 2 hours, then take it out and ventilate for 6 hours.

[0069] S3. Germination: The soaked bitter buckwheat was subjected to intermittent culture treatment under constant temperature and humidity conditions of 25°C and 70% humidity, and 3% sucrose solution was sprayed every 2 hours, and germinated for 4 days to obtain sprouted bitter buckwheat. The intermittent culture treatment process is: the soaked bitter buckwheat was exposed to light for 12 hours and then treated in darkness for 12 hours.

[0070] S4. Washing and beating: Wash the buckwheat sprouts with clean water, and add drinking water to the washed buckwheat sprouts at a weight ratio of 1:15 for beating, and filter.

[0071] S5. Drying: The filtrate was concentrated and spray-dried, wherein the air inlet temperature was 170°C, the air outlet temperature was 80°C, the atomizer speed was 15000 r / min, and the feed rate was 70 mL / min.

[0072] Among them, the nutritional components of bitter buckwheat before and after germination were determined, and the nutritional content is shown in Table 1:

[0073] Table 1 Nutritional content of buckwheat flour before and after germination

[0074]

[0075] As shown in Table 1, the dietary fiber, total flavonoids and total phenol contents in tartary buckwheat at germination day 3 were higher than those before germination, the protein and starch contents decreased by 0.36% and 5.52%, respectively, and the total flavonoids content increased from 1.67% to 3.84%, which indicates that the starch stored in the grains during germination of tartary buckwheat may produce phenylalanine through the shikimic acid pathway, and then synthesize flavonoids in large quantities through the phenylpropanoid metabolic pathway.

[0076] Example 2: Protein hydrolysis of tartary buckwheat after germination

[0077] (1) Virtual hydrolysis: The amino acid sequences of the main proteins in buckwheat were collected, including 2S seed storagealbumin protein, BW10KD allergen protein, 13S globulin seed storage protein, Trypsin inhibitor, and Maturase K. Papain (EC3.4.22.2), alkaline protease (EC3.4.21.62), trypsin (EC3.4.21.4), bromelain (EC3.4.22.32), and double enzymes were used. A total of 10 enzyme treatment methods were used to virtually hydrolyze the above proteins, and the theoretical hydrolysis degree and DPP-IV inhibitory activity (frequency) were calculated. The results are shown in Table 2.

[0078] Table 2 Virtual hydrolysis prediction

[0079]

[0080] Note: 1 is papain, 2 is alkaline protease, 3 is trypsin, and 4 is bromelain.

[0081] The results showed that compared with other groups, the hydrolysis of buckwheat protein using papain and alkaline protease (1+2 group) could obtain more DPP-IV inhibitory active peptides.

[0082] (2) In vitro hydrolysis: The best double enzyme (1+2 group) and the corresponding single enzyme in (1) were used to hydrolyze the budding buckwheat flour. The buckwheat flour and water liquid ratio was 1:20 (m / v) and heated at 100℃ for 10 min to completely denature the protein, with continuous stirring during the process. After cooling to room temperature, 1 mol / L NaOH or HCl was used to adjust the solution to the optimal pH value for the protease; protease was added according to the enzyme substrate ratio: 9000U / g protein for hydrolysis; after the hydrolysis was completed, the hydrolysis reaction was terminated by heating in a boiling water bath. The degree of hydrolysis was determined by formaldehyde titration. After the enzymatic hydrolyzate was diluted 50 times, the DPP-IV inhibitory activity was determined using a DPP-IV ELISA kit. The hydrolysis conditions are shown in Table 3.

[0083] Table 3 Hydrolysis conditions

[0084]

[0085]

[0086] The in vitro hydrolysis results are shown in Table 4:

[0087] Table 4 In vitro hydrolysis degree and DPP-IV inhibitory activity

[0088]

[0089] The hydrolysis degree of the filtrate homogenate treated with papain + alkaline protease was the highest, which means more small molecule peptides were obtained. The virtual hydrolysis results were consistent with the actual hydrolysis results, showing that the combined use of two enzymes can obtain higher DPP-IV inhibitory activity.

[0090] Example 3: Screening and verification of DPP-IV inhibitory peptides from the enzyme-treated solution of germinating bitter buckwheat homogenate

[0091] The peptide sequences in the samples were analyzed by LC-MS / MS, and a total of 5426 peptides were identified.

[0092] The screening conditions for DPP-IV inhibitory peptides are:

[0093] (1) Use PeptideRanker to predict the biological activity of peptides;

[0094] (2) Toxicity prediction using Toxinpred;

[0095] (3) Allergenicity prediction using AllerTOP;

[0096] (4) Select sequences with a peptide chain length of <6, a credibility ranking in the top 100, an activity score >0.95, and no toxicity or allergenicity.

[0097] Molecular docking was used for secondary screening, and all peptides were subjected to energy minimization and hydrogenation treatment, and the 3D structure of DPP-IV was obtained from the PDB database (PDB ID: 4N8D).

[0098] like Figure 2 As shown in Table 5, the peptides were docked after the removal of solvent and organic matter. Auto Dock was used for molecular docking and screening was performed according to the docking energy value. The top 10 peptides with the lowest docking energy values ​​were taken, and after removing the DPP-IV inhibitory peptides reported in the BIOPEP-UWM database, the remaining 3 peptides were synthesized by solid phase for subsequent activity verification. The molecular docking binding energy is shown in Table 5.

[0099] Table 5 Docking binding energy

[0100]

[0101] (3) The docking results are as follows Figure 3As shown, PIW, IF, and CPPL form hydrogen bonding forces with amino acid residues such as TYR666, TYR662, and GLU206 in the active pocket of the receptor protein, which is the key interaction force for the three polypeptides to have DPP-IV inhibitory activity.

[0102] Example 4: Verification of DPP-IV inhibitory activity of PIW, IF, and CPPL

[0103] After solid phase synthesis, peptides PIW, IF, and CPPL were tested for activity using a DPP-IV ELISA kit. The results are shown in Table 6.

[0104] Table 6 DPP-IV inhibitory peptide activity test results

[0105]

[0106] The results showed that CPPL, IF, and PIW all had strong DPP-IV inhibitory activity. 50 All of them were less than 100 μM. Among them, PIW had the strongest DPP-IV inhibitory activity (IC 50 is 25.27 μM).

[0107] Example 5: Evaluation of in vitro synergistic hypoglycemic ability

[0108] The in vitro hypoglycemic activities (2 mg / mL) of the filtered pulp before germination (A), the filtered pulp after germination (B), and the enzymatic hydrolyzate of the filtered pulp after germination (C) were compared, including the inhibitory effects on α-glucosidase, α-amylase, and DPP-IV. The test results are shown in Table 7.

[0109] Table 7 In vitro hypoglycemic effect of different treatments of tartary buckwheat filtrate

[0110]

[0111] The results showed that the untreated tartary buckwheat filtrate had a certain degree of inhibitory effect on the three enzymes. Compared with the germination (A), the inhibitory effect of α-glucosidase and α-amylase in the tartary buckwheat filtrate (B) after germination was significantly increased, which was attributed to the increase in flavonoid content after germination, but it had almost no effect on the activity of DPP-IV. However, after germination and enzymatic hydrolysis of tartary buckwheat (C), the inhibition rate of DPP-IV activity increased by 2.01 times compared with the germination treatment alone (B), reaching 80.84±0.30% (p<0.05), indicating that after protease treatment, the small molecular peptides released showed DPP-IV inhibitory activity. Enzymatic hydrolysis did not significantly increase the inhibitory activity of α-glucosidase and α-amylase in the filtrate, indicating that the germination and enzymatic hydrolysis of tartary buckwheat, the increased flavonoid content and the free DPP-IV inhibitory peptides can achieve a synergistic hypoglycemic effect by reducing glucose intake and increasing glucose consumption, respectively.

[0112] Example 6: Evaluation of in vivo hypoglycemic ability

[0113] The acute hypoglycemic activity of the pre-germination filtrate (A), post-germination filtrate (B), and post-germination filtrate enzymatic hydrolyzate (C) of Tartary buckwheat was compared by oral glucose tolerance test (OGTT). Four-week-old male C57BL / 6 mice (n=6 / group) were fasted for 12 h 30 min before oral glucose load (2 g / kg BW) by gavage. Blood glucose levels were measured by caudal vein blood sampling at 0, 30, 60, and 120 min after administration. The area under the curve increment (AUC) at 120 min was calculated for each animal using the trapezoidal rule.

[0114] Table 8 Results of oral glucose tolerance test in different treatment groups

[0115]

[0116] The results showed that the fasting blood glucose levels (0-120min) of each group changed in a time-dependent manner after oral administration of glucose solution. Compared with the blank group Con, the AUC 120 min Compared with the Con group (blank group), the blood sugar level was significantly reduced by 3.68% and 5.22%, respectively. At the same dosage, the enzymatic hydrolysis of tartary buckwheat germination (group C) showed more significant hypoglycemic activity, AUC 120 min In addition, the drug took effect in all groups within 30 minutes of taking the drug, achieving a significant blood sugar-lowering effect.

[0117] In summary, it can be seen from the data in Table 1 that the dietary fiber, total flavonoids and total phenolic contents in the germination 3d bitter buckwheat in Example 1 are higher than those before germination; in Example 2, it can be seen from the data in the table that the hydrolysis degree of the filtrate homogenate treated with dual enzymes is the highest, which means that more small molecule peptides are obtained. The virtual hydrolysis results are consistent with the actual hydrolysis results, showing that the dual enzyme combination has a higher DPP-IV inhibitory activity, which means that the virtual hydrolysis has a reference and guiding significance in the selection of actual hydrolysis conditions, simplifying the exploration of protein hydrolysis process conditions.

[0118] In Example 3, Figure 3 It can be seen that CPPL, IF, PIW form hydrogen bonding forces with amino acid residues such as TYR666, TYR662, GLU206 in the active pocket of the receptor protein, which is the key interaction force for the three polypeptides to have DPP-IV inhibitory activity; the data in Example 4 show that the three polypeptides (CPPL, IF, PIW) all have strong DPP-IV inhibitory activity, IC 50 All of them were less than 100 μM. Among them, PIW had the strongest DPP-IV inhibitory activity (IC 50 is 25.27 μM).

[0119] In Example 5, the results of in vitro hypoglycemic treatment showed that the increase in flavonoid content after tartary buckwheat germination can significantly inhibit the activity of α-glucosidase and α-amylase, but has no significant inhibition on DPP-IV activity. After tartary buckwheat germination and then enzymatic treatment, the free small peptides have a significant inhibitory effect on DPP-IV, but have no significant inhibition on α-glucosidase and α-amylase activities. It shows that the increased flavonoid content and the free DPP-IV inhibitory peptides can achieve a synergistic hypoglycemic effect by reducing glucose intake and increasing glucose consumption, respectively.

[0120] In Example 6, at the same dosage, after germination and enzymatic hydrolysis, tartary buckwheat showed significant hypoglycemic activity, AUC 120 min Compared with the blank group, it decreased by 13.91%, which was better than the pre-germination and post-germination treatment groups. And the hypoglycemic effect in the body can be exerted within 30 minutes of taking the medicine.

[0121] By performing docking scoring, activity, toxicity and physicochemical property comprehensive scoring on the peptides identified by mass spectrometry, three new peptides with the strongest DPP-IV inhibitory activity were obtained, namely CPPL, IF and PIW; this application is guided by the computer virtual hydrolysis results, simplifies the in vitro enzymatic hydrolysis conditions, and efficiently and accurately prepares buckwheat flour rich in DPP-IV inhibitory peptides; the buckwheat flour in this application is treated with germination treatment and enzymatic hydrolysis technology to achieve efficient conversion of flavonoids while maximizing the content of DPP-IV inhibitory peptides in the buckwheat flour, effectively synergistically lowering blood sugar, and realizing the scientific development of buckwheat blood sugar-lowering products, which has great application value for the improvement or treatment of type 2 diabetes.

[0122] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for preparing synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides, characterized in that: The following steps are involved: S1. Ultrasonic pretreatment: Wash and dry the bitter buckwheat, sterilize it and then perform ultrasonic treatment; S2. Soaking the buckwheat: placing the tartary buckwheat after ultrasonic pretreatment at a constant temperature of 25±5℃ for two intermittent soaking treatments; S3. Germination: The soaked bitter buckwheat was intermittently cultured under constant temperature and humidity conditions, sprayed with 3-6% sucrose solution every 2-4 hours, and germinated for 2-4 days to obtain germinated bitter buckwheat; S4. Washing and beating: After washing the buckwheat buds with clean water, adding drinking water at a weight ratio of 1:10-1:15 for beating, and filtering to prepare a filtered slurry; S5. Virtual hydrolysis: Collect the amino acid sequences of buckwheat proteins, use single enzyme or multiple enzymes to virtually hydrolyze the sequences through the database and predict the DPP-IV inhibitory activity of the resulting peptides; S6. In vitro hydrolysis: According to the guiding parameters obtained in S5, the filtrate obtained in S4 is heated, cooled to room temperature, the solution is adjusted to the optimal pH value of the protease, and the protease is added according to the enzyme-substrate ratio and then hydrolyzed; S7. Sequence identification: After the enzymatic hydrolysate cooled to room temperature is centrifuged, the supernatant is freeze-dried for peptide sequence identification; S8. DPP-IV inhibitory peptide screening: Based on the identification results of S7, a series of screening conditions are used to screen the obtained peptides step by step to obtain specific peptides with high DPP-IV inhibitory activity; S9. Drying: The enzymatically hydrolyzed liquid is concentrated and spray-dried, or dried using other techniques such as freeze-drying.

2. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 1, characterized in that: The specific steps of virtual hydrolysis in S5 are as follows: collecting the amino acid sequence of tartary buckwheat protein, using the BIOPEP-UWM database to virtually hydrolyze the sequence and predict the DPP-IV activity of the obtained peptide.

3. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 1, characterized in that: The specific steps of in vitro hydrolysis in S6 are as follows: heating the filtered slurry obtained in S4 at 90-100°C for 10-15 minutes, stirring continuously during the period to ensure that the protein is completely denatured by heat; after cooling to room temperature, adjusting the solution to the optimal pH value of the protease using NaOH or HCl; adding protease according to the enzyme-substrate ratio: 6000-10000U / g protein for hydrolysis for 4-8 hours; after the hydrolysis is completed, heating in a boiling water bath to terminate the hydrolysis reaction.

4. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 1, characterized in that: The specific steps of sequence identification in S7 are as follows: take 20-30 mL of the enzymatic hydrolysate cooled to room temperature, centrifuge at 6000-8000 rpm for 15-20 min, take the supernatant for freeze-drying, and perform sequence identification using LC-MS / MS.

5. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 1, characterized in that: The screening conditions in S8 are specifically molecular docking, activity prediction, physicochemical property prediction, sensitization and toxicity prediction, and amino acid composition distribution.

6. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 1, characterized in that: The ultrasonic pretreatment in S1 comprises the following steps: (1) Optimization and cleaning: Select high-quality bitter buckwheat produced in the current year, select those with full grains for later use, and clean and dry them; (2) Disinfection: Soak the treated buckwheat in a 5% to 8% sodium hypochlorite solution for 10 to 15 minutes; (3) Ultrasonic pretreatment: The sterilized buckwheat was placed under ultrasound with a power of 25 kHz for 12-16 min.

7. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 1, characterized in that: The specific process of the intermittent soaking treatment in S2 is: soak the bitter buckwheat in a 3% sucrose solution for 2-4 hours and then take it out for ventilation for 6-9 hours; the intermittent culture treatment process in S3 is: expose the bitter buckwheat to light for 12 hours and then keep it in the dark for 12 hours.

8. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 2, characterized in that: The protease in S5 is selected from one or more of alkaline protease, papain, neutral protease, bromelain and trypsin.

9. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 3, characterized in that: The hydrolysis conditions selected in S6 are performed and determined based on the virtual hydrolysis results of S5, and the degree of hydrolysis is determined by formaldehyde titration.

10. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 1, characterized in that: In the S9, the spray drying air inlet temperature is 170±10°C, the air outlet temperature is 80±5°C, the atomizer speed is 15000r / min, and the feed rate is 70±5mL / min.

11. The method for preparing the synergistic hypoglycemic buckwheat flour rich in flavonoids and DPP-IV inhibitory peptides according to claim 1, characterized in that: The DPP-IV activity inhibitory peptide sequences in the S8 are CPPL, IF, and PIW.

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