Chickpea protein peptide with hypoglycemic activity and preparation method and application thereof

Abstract

The invention provides chickpea protein peptide as well as a preparation method and application thereof. The preparation method of the chickpea protein peptide provided by the invention comprises the following steps: sequentially carrying out enzymolysis on chickpea protein isolate by using neutral protease, pepsin and pancreatin; after the enzymolysis is finished, performing ultrafiltration on an enzymolysis product by using an ultrafiltration membrane with the interception amount of 3kDa, and collecting components with the molecular weight of less than 3kDa; and finally, carrying out gel chromatography on the component, and collecting the corresponding component to obtain the chickpea protein peptide. The chickpea protein peptide prepared by the method has inhibitory activity on DPP-IV, alpha-glucosidase and alpha-amylase, can effectively inhibit the activity of Caco-2 cell sucrase, maltase and DPP-IV, and has a certain inhibitory effect on glucose transport; therefore, the chickpea protein peptide provided by the invention has good hypoglycemic activity, and a new thought is provided for development of food-borne functional protein peptides.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a chickpea protein peptide with blood sugar-lowering activity, and a preparation method and application thereof. Background Art

[0002] Diabetes is a chronic disease characterized by hyperglycemia, mainly caused by insufficient insulin secretion or utilization disorders. Patients often suffer from multiple serious complications, such as cardiovascular disease, retinopathy, nephropathy, etc., which seriously affect the quality of life and health of patients. According to data from The Lancet, approximately 828 million people in the world suffered from diabetes in 2022. The International Diabetes Federation predicts that the number of people with diabetes worldwide will increase to 643 million by 2030 and reach 783 million by 2045. Diabetes has become a serious public health problem worldwide, and there is an urgent need to develop safe and effective blood sugar-lowering strategies. Dipeptidyl peptidase-4 (DPP-IV), α-amylase and α-glucosidase are important targets for regulating blood sugar balance. DPP-IV is a plasma membrane-bound serine protease that primarily degrades glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), thereby reducing insulin secretion and increasing glucagon release, leading to elevated blood glucose levels. DPP-IV inhibitors can block this process, reducing the degradation of GLP-1 and GIP and delaying the rise in blood glucose levels. α-Amylase and α-glucosidase are digestive enzymes that catalyze the breakdown of polysaccharides into monosaccharides, enabling their absorption in the small intestine and triggering a rapid rise in blood glucose levels after a meal. α-Amylase and α-glucosidase inhibitors inhibit the activity of these enzymes, slowing glucose release and absorption, thereby effectively controlling postprandial blood glucose fluctuations. Commonly used inhibitors in clinical treatment include sitagliptin and acarbose, but these drugs are often associated with adverse reactions such as gastrointestinal discomfort, lower limb edema, and liver dysfunction. In recent years, food-derived functional protein peptides have attracted considerable attention due to their safety, ease of absorption, and low production costs. At present, bitter melon peptides launched by Atomy and Amgen have been widely used in functional foods, adjuvant treatment of diabetes and other fields. Diprotin A (IPI) is the most active DPP-IV inhibitory peptide (IC 50 value is 1.62 μM), but has not yet been commercialized.

[0003] The main methods for preparing food-derived functional protein peptides include protease hydrolysis, chemical synthesis, and microbial fermentation. Among them, protease hydrolysis has the advantages of mild reaction conditions, few side reactions, and high product safety, and is currently the main method for preparing functional protein peptides. A large number of studies have used protease hydrolysis of plant and animal proteins to prepare DPP-IV, α-amylase, and α-glucosidase inhibitory peptides, IC 50 The values were 0.01-3.89 mg / mL, but their activity was much lower than that of inhibitors such as sitagliptin and acarbose (IC 50 The value is 0.0075-52.2μg / mL). In recent years, the discovery of new microbial proteases and the pretreatment of protein raw materials have provided new ideas for improving the hypoglycemic activity. The use of ultrasound, pH shift-assisted hydrolysis and the combination of new microbial proteases with commercial proteases can effectively improve the DPP-IV and α-amylase inhibitory activity (IC 50 Serine proteases and metalloproteases derived from Bacillus subtilis hydrolyze soy protein isolate, wheat gluten, and casein, yielding hydrolyzates with DPP-IV, α-amylase, and α-glucosidase inhibition rates of 34.8%, 33.98%, and 48.33%, respectively. However, existing research has primarily focused on the preparation of protein peptides with a single hypoglycemic activity. However, actual blood sugar regulation involves multiple signaling pathways and the synergistic effects of multiple bioactive factors, making it difficult for a single activity to achieve the desired effect. Currently, there are no reports of food-derived functional protein peptides that simultaneously inhibit DPP-IV, α-amylase, and α-glucosidase. Summary of the Invention

[0004] The present invention provides a chickpea protein peptide and a preparation method and application thereof. The chickpea protein peptide has DPP-IV, α-amylase and α-glucosidase inhibitory activities and has blood sugar lowering activity.

[0005] In a first aspect, the present invention provides a method for preparing chickpea protein peptide, comprising:

[0006] S1. Obtaining chickpea protein isolate, and mixing the chickpea protein isolate with deionized water to prepare a protein suspension;

[0007] S2, adding neutral protease, pepsin and pancreatin to the protein suspension in sequence to enzymolyze the protein in sequence, and collecting the enzymolysis products after the enzymolysis is completed; the neutral protease is derived from Aspergillus oryzae 3.042;

[0008] S3, ultrafiltration of the enzymatic hydrolysate using an ultrafiltration membrane with a cut-off of 3 kDa to collect components with a molecular weight less than 3 kDa;

[0009] S4. Use a Sephadex G-15 chromatography column to perform gel chromatography on the components with a molecular weight less than 3 kDa, the eluent is a phosphate buffer, the concentration of the phosphate buffer is 10 mmol / L, the pH is 7.0, the ultraviolet detection wavelength is 280 nm, the elution rate is 0.8 mL / min, and the components flowing out with an elution time of 160-191 min are collected, and the chickpea protein peptide is obtained after drying.

[0010] Preparation method as described above, in S1, the chickpea protein isolate refers to a plant protein derived from chickpea (Cicerarietinum L.), which can be prepared by isoelectric precipitation method, salting-out method, and alkali-soluble acid precipitation method. In a specific embodiment, the chickpea protein isolate is prepared by alkali-soluble acid precipitation method, specifically comprising the steps of: grinding chickpea into powder to obtain chickpea flour; chickpea flour and deionized water are mixed in a ratio of 1: 10 (w / v), and the pH is adjusted to 10.5 using 1mol / L sodium hydroxide solution, and dissolved at room temperature (500rpm) for 2h. The mixed solution is centrifuged at 10000r / min for 5min, and the supernatant is collected. The pH of the supernatant is adjusted to 4.5 using 1mol / L hydrochloric acid, stirred and dissolved at room temperature for 1h, and the precipitate is collected after centrifugation, and it is freeze-dried to obtain chickpea protein isolate.

[0011] In the preparation method described above, the ratio of the mass of the chickpea protein isolate to the volume of deionized water is 1:(10-30).

[0012] According to the preparation method described above, chickpea protein isolate and deionized water are fully mixed under stirring for 2-4 hours at a stirring rate of 150-250 rpm.

[0013] In the preparation method described above, the pH of the protein suspension is adjusted to 6.0-8.0 to provide a suitable pH environment for the subsequent enzymatic hydrolysis process. In a specific embodiment, the pH of the protein suspension can be adjusted using 1 mol / L hydrochloric acid solution or 1 mol / L sodium hydroxide solution.

[0014] In the preparation method described above, the neutral protease is derived from Aspergillus oryzae 3.042, which is numbered txid1160506 in NCBI. The coding sequence of the neutral protease derived from Aspergillus oryzae 3.042 is recorded in the supplementary data of the following document: Ke Y, Huang WQ, Li JZ, et al. Enzymatic Characteristics of a Recombinant Neutral Protease I (rNpI) from Aspergillus oryzae Expressed in Pichia pastoris [J]. Journal of Agricultural and Food Chemistry, 2012, 60(49): 12164-12169.

[0015] The preparation method as described above, based on each gram of chickpea protein isolate, the addition amount of the neutral protease is 1000-2000U; specifically, it can be 1000U, 1050U, 1100U, 1150U, 1200U, 1250U, 1300U, 1350U, 1400U, 1450U, 1500U, 1550U, 1600U, 1650U, 1700U, 1750U, 1800U, 1850U, 1900U, 1950U, 2000U or within the range of any two thereof. Further, based on each gram of chickpea protein isolate, the addition amount of the neutral protease is 1500U. The neutral protease activity unit (U) is defined as: hydrolyzing casein to produce 1 μg of tyrosine per minute at 55°C, defined as one protease activity unit (U).

[0016] In the preparation method described above, during the enzymatic hydrolysis process of the neutral protease, the pH of the reaction system is adjusted to 7.0-9.0, specifically 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or within the range of any two thereof. Further, during the enzymatic hydrolysis process of the neutral protease, the pH of the reaction system is adjusted to 8.0.

[0017] In the preparation method described above, the enzymatic hydrolysis time of the neutral protease is 1-2 hours, specifically 1.0 hour, 1.1 hour, 1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2.0 hours, or any two thereof. Further, the enzymatic hydrolysis time of the neutral protease is 1 hour.

[0018] According to the preparation method described above, after the enzymatic hydrolysis of the neutral protease is completed, the reaction system is placed in boiling water to inactivate the neutral protease in a water bath, and after cooling to room temperature, the subsequent enzymatic hydrolysis treatment is continued.

[0019] According to the preparation method described above, the CAS number of the pepsin is 9001-75-6.

[0020] Preparation method as described above, based on every gram of chickpea protein isolate, the addition amount of described pepsin is 1000-2000U, specifically can be 1000U, 1050U, 1100U, 1150U, 1200U, 1250U, 1300U, 1350U, 1400U, 1450U, 1500U, 1550U, 1600U, 1650U, 1700U, 1750U, 1800U, 1850U, 1900U, 1950U, 2000U or within the scope of any two compositions therein. Further, based on every gram of chickpea protein isolate, the addition amount of described pepsin is 2000U. Described pepsin activity unit (U) is defined as: at 37 DEG C, hydrolyze casein per minute and produce 1 μg tyrosine, defined as one protease activity unit (U).

[0021] In the preparation method described above, during the pepsin hydrolysis process, the pH of the reaction system is adjusted to 1.5-3.0, specifically 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, or any two thereof. Furthermore, during the pepsin hydrolysis process, the pH of the reaction system is adjusted to 2.0.

[0022] In the preparation method described above, the enzymatic hydrolysis time of the pepsin is 1-2 hours, specifically 1.0 hour, 1.1 hour, 1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2.0 hours, or any two thereof. Further, the enzymatic hydrolysis time of the pepsin is 2 hours.

[0023] According to the preparation method described above, the CAS number of the pancreatin is 8049-47-6.

[0024] Preparation method as described above, based on every gram of chickpea protein isolate, the addition amount of described pancreatin is 1000-2000U, specifically can be 1000U, 1050U, 1100U, 1150U, 1200U, 1250U, 1300U, 1350U, 1400U, 1450U, 1500U, 1550U, 1600U, 1650U, 1700U, 1750U, 1800U, 1850U, 1900U, 1950U, 2000U or within the range of any two compositions therein. Further, based on every gram of chickpea protein isolate, the addition amount of described pancreatin is 2000U. The definition of described pancreatic enzyme activity unit (U) is: at 37 DEG C, hydrolyze casein per minute to produce 1 μg of tyrosine, defined as one protease activity unit (U).

[0025] In the preparation method described above, during the pancreatic enzymatic hydrolysis, the pH of the reaction system is adjusted to 6.0-8.0, specifically 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or any two thereof. Further, during the pancreatic enzymatic hydrolysis, the pH of the reaction system is adjusted to 7.0.

[0026] In the preparation method described above, the enzymatic hydrolysis time of the pancreatic enzyme is 1-2 hours, specifically 1.0 hour, 1.1 hour, 1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2.0 hours, or any two thereof. Furthermore, the enzymatic hydrolysis time of the pepsin is 2 hours.

[0027] In the preparation method described above, the temperature of the enzymatic hydrolysis is 35°C-60°C, specifically 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C or within the range of any two thereof.

[0028] According to the preparation method described above, after the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is centrifuged at a speed of 8000-12000 r / min for 5-15 min, and the supernatant is collected for ultrafiltration separation to collect components with a molecular weight less than 3 kDa.

[0029] According to the preparation method described above, the components with a molecular weight of less than 3 kDa collected by ultrafiltration are subjected to gel chromatography, wherein the gel chromatography uses a Sephadex G-15 chromatography column (1000 mm × 12 mm), the sample volume is 2 mL, a phosphate buffer with a pH of 7.0 and a concentration of 10 mmol / L is used as the eluent, the ultraviolet detection wavelength is 280 nm, the elution rate is 0.8 mL / min, and elution is performed according to the above-mentioned isocratic elution conditions. The components flowing out at an elution time of 160-191 min are collected, and the chickpea protein peptide is obtained after drying.

[0030] In a second aspect, the present invention provides a chickpea protein peptide prepared according to any of the above-mentioned preparation methods.

[0031] The chickpea protein peptide as described above may be a mixture of at least two peptide segments, or may be a functional peptide segment with specific functions extracted from the mixture.

[0032] The chickpea protein peptide as described above has DPP-IV inhibitory activity, α-glucosidase inhibitory activity and α-amylase inhibitory activity. Furthermore, the chickpea protein peptide has a half inhibitory concentration (IC50) of DPP-IV in vitro. 50 ) was 0.09 mg / mL, and the half-inhibitory concentration (IC 50 ) was 0.12 mg / mL, and the half-inhibitory concentration (IC 50 ) is 0.06 mg / mL.

[0033] As described above, the chickpea protein peptide has an inhibition rate of 19.33%, 37.63% and 42.22% on sucrase on Caco-2 cells, respectively, when the concentration of the chickpea protein peptide is 0.05, 0.10 and 0.20 mg / mL, and an inhibition rate of 22.42%, 44.34% and 51.44% on maltase on Caco-2 cells, respectively. There is a dose-dependent relationship between the inhibition rate and the concentration of the chickpea protein peptide.

[0034] The chickpea protein peptides mentioned above, at the chickpea protein peptide concentration of 0.10 and 0.20 mg / mL, showed inhibitory activity against DPP-IV on Caco-2 cells by reducing the luminescence signal to 2.84×10 5 RLU and 1.86×10 5 RLU.

[0035] For the chickpea protein peptide as described above, when the concentration of chickpea protein peptide is 0.05, 0.10 and 0.20 mg / mL, when the glucose concentration is 25 mM, the glucose transport inhibition rates are 13.73%, 26.47% and 50.65%, respectively; when the glucose concentration is 5 mM, the glucose transport inhibition rates are 17.14%, 38.10% and 52.38%, respectively. There is a dose-dependent relationship between the inhibition rate and the concentration of chickpea protein peptide, and there is no significant difference between the glucose transport inhibition rate at high glucose levels and the glucose transport inhibition rate at low glucose levels.

[0036] The chickpea protein peptides described above include at least 22 peptide segments, including FGL, GLSVFVPLNL, SFGW, SGW, YGM, YGW, YWM, EDW, ELW, FEGW, FGY, FPW, FSW, GCFSCNSGTG, GFGP, LGW, LLGGF, VGF, VPW, WSFG, WVP and YGF; among them, according to the AntiT2DMP-Pred tool (https: / / babalab-skku.org / AntiT2 DMP-Pred / ) analyzed the confidence levels of the hypoglycemic activity of the above peptides. The confidence levels of the first seven peptides (FGL, GLSVFVPLNL, SFGW, SGW, YGM, YGW, and YWM) were higher than 0.9, and the confidence levels of the last 15 peptides (EDW, ELW, FEGW, FGY, FPW, FSW, GCFSCNSGTG, GFGP, LGW, LLGGF, VGF, VPW, WSFG, WVP, and YGF) were between 0.8 and 0.9.

[0037] In a third aspect, the present invention provides a use of any of the above-mentioned chickpea protein peptides in the preparation of a product with blood sugar lowering function.

[0038] In the application described above, the product may be at least one of food, health care product, and medicine.

[0039] It is understood that the product includes the chickpea protein peptide and may also include other active ingredients and / or inactive ingredients.

[0040] The chickpea protein peptide prepared according to the method provided by the present invention has inhibitory activity against DPP-IV, α-glucosidase and α-amylase, can effectively inhibit the activity of Caco-2 cell sucrase, maltase and DPP-IV, and has a certain inhibitory effect on glucose transport. Therefore, it has good hypoglycemic activity and provides a new idea for the development of food-derived functional protein peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1The IC values of chickpea protein hydrolysate after ultrafiltration with different ultrafiltration membranes for three enzymes 50 Test results;

[0042] Figure 2 This is the peak diagram of the component with molecular weight less than 3kDa after ultrafiltration of chickpea protein hydrolysate during gel chromatography;

[0043] Figure 3 IC values of chickpea protein peptides on three enzymes 50 Test results;

[0044] Figure 4 is the survival rate of Caco-2 cells cultured with different concentrations of chickpea protein peptides;

[0045] Figure 5 The luminescence signal intensity test results of different concentrations of chickpea protein peptides inhibiting the secretion of DPP-IV in Caco-2 cells;

[0046] Figure 6 The results show the inhibition rate of different concentrations of chickpea protein peptide on the secretion of maltase in Caco-2 cells;

[0047] Figure 7 The results show the inhibition rate of chickpea protein peptides at different concentrations on the secretion of sucrase by Caco-2 cells;

[0048] Figure 8 Effects of different concentrations of chickpea protein peptides on glucose transport in Caco-2 monolayer cells. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0050] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0051] The Aspergillus oryzae neutral protease used in the following examples is a recombinant protease expressed by cloning, and the specific steps are as follows:

[0052] 1. Obtaining gene fragments: According to the supplementary data of the following literature: Ke Y, Huang WQ, Li JZ, et al. Enzymatic Characteristics of a Recombinant Neutral Protease I (rNpI) from Aspergillus oryzae Expressed in Pichia pastoris [J]. Journal of Agricultural and Food Chemistry, 2012, 60(49): 12164-12169, the gene fragment encoding neutral protease AoNPI was synthesized.

[0053] 2. Construction of recombinant vector and strain: The above gene fragments were ligated to the pPIC9K vector to obtain the recombinant plasmid pPIC9K-AoNPI. The correctly sequenced recombinant plasmid was linearized with Sac I and transformed into Pichia pastoris GS115. The recombinant Pichia pastoris strain was obtained by high copy screening.

[0054] 3. Fermentation to obtain Aspergillus oryzae neutral protease:

[0055] (1) Seed liquid culture: The recombinant strain was inoculated into YPD liquid medium and cultured at 30°C and 200 rpm with shaking until the OD 600 The value is about 10.0, and the seed solution is obtained.

[0056] (2) Fed-batch fermentation: The seed solution was inoculated into the fermentation medium at a 10% inoculum rate, sterilized, the pH was adjusted, PTM1 was added, and fermentation was carried out at 30°C. After the glycerol was exhausted, 50% glycerol was fed. The feeding was stopped when the wet weight of the cells reached 180 g / L, the pH was adjusted to 6.0, and the cells were starved for 30 min. 100% methanol was fed to induce enzyme production. After 6-7 days of fermentation, the cells were centrifuged at 10,000 rpm for 10 min, and the supernatant was collected to obtain a crude Aspergillus oryzae neutral protease solution (the crude enzyme solution only contained Aspergillus oryzae neutral protease).

[0057] The pepsin used in the following examples was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with the product number S10027; and the pancreatin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. with the product number P7545.

[0058] The Caco-2 cell line used in the following examples was obtained from the Cell Bank of the Chinese Academy of Sciences.

[0059] Example 1. Selection of protease for chickpea protein separation and hydrolysis

[0060] 1. Prepare chickpea protein isolate: Grind chickpeas into powder, mix chickpea flour with deionized water at a ratio of 1:10 (w / v). Use 1mol / L sodium hydroxide solution to adjust the pH to 10.5, stir (500rpm) at room temperature and dissolve for 2h. Centrifuge the mixed solution at 10000r / min for 5min, and collect the supernatant. Adjust the pH of the supernatant to 4.5 using 1mol / L hydrochloric acid, stir and dissolve at room temperature for 1h, collect the precipitate after centrifugation, and freeze-dry it to obtain chickpea protein isolate.

[0061] 2. Enzymolysis: Chickpea protein was enzymolyzed using the proteases in the order shown in Table 1. The enzymolysis conditions for each enzymolysis were the optimal conditions for the corresponding proteases. The enzymolysis time for pepsin and pancreatin was 2 hours, and the enzyme addition amount was 2000 U / g. The remaining proteases were enzymolyzed for 1 hour, and the enzyme addition amount was 1500 U / g. After each enzymolysis, the protease was inactivated in a boiling water bath for 5 minutes. After all the protease enzymolysis was completed, the chickpea protein hydrolyzate was obtained by centrifugation. The inhibition rates of different enzymolysis products on DPP-IV, α-glucosidase, and α-amylase were determined, and the results are shown in Table 1.

[0062] The DPP-IV inhibition rate was determined as follows: chickpea protein hydrolysate was dissolved in deionized water to prepare a sample solution with a protein concentration of 2 mg / mL. 25 μL of the sample solution was mixed with 25 μL of a 1.6 mM Gly-Pro-pNA solution, preheated at 37°C for 10 min, and 50 μL of a DPP-IV solution (8 U / L, dissolved in 100 mM Tris-HCl buffer, pH 8.0) was added. The reaction was allowed to proceed at 37°C for 60 min, and 100 μL of a 1 M HAc-NaAc buffer (pH 4.0) was added to terminate the reaction. The absorbance was measured at 405 nm. Tris-HCl buffer was used instead of the DPP-IV solution as a sample blank control, deionized water was used instead of the sample as a negative control, and Tris-HCl buffer was used instead of the DPP-IV solution and deionized water was used instead of the sample as a negative blank control. The DPP-IV inhibition rate was calculated as follows:

[0063] DPP-IV inhibition rate (%) = [1- (As-Ase) / (Ac-Ace)] × 100 Formula 1

[0064] In formula 1: As represents the absorbance value of the sample; Ase represents the absorbance value of the sample blank control; Ac represents the absorbance value of the negative control; Ace represents the absorbance value of the negative blank control.

[0065] The chickpea protein peptide solution was diluted to different concentrations and the DPP-IV inhibition rate was determined. The DPP-IV inhibition rate was plotted as the ordinate and the log value of the sample concentration was plotted as the abscissa. GraphPad Prism 8.0 was used to perform nonlinear fitting to calculate the half inhibitory concentration (IC50) of the sample. 50 ).

[0066] Among them, the method for α-glucosidase inhibition rate is as follows: dissolve chickpea protein peptide in deionized water to prepare a sample solution with a protein concentration of 0.5 mg / mL. Take 100 μL of sample solution and mix it with 50 μL of p-nitrophenyl-α-D-pyranoglucoside (pNPG) solution (2 mM), preheat at 37 ° C for 10 min, add 50 μL of α-glucosidase solution (0.5 U / mL, dissolved in 0.1 M potassium phosphate buffer, pH 6.9), react at 37 ° C for 10 min, add 100 μL of sodium carbonate solution (0.2 M) to terminate the reaction, and measure the absorbance at 405 nm. Potassium phosphate buffer is used instead of α-glucosidase solution as a sample blank control, deionized water is used instead of sample as a negative control, potassium phosphate buffer is used instead of α-glucosidase solution, and deionized water is used instead of sample as a negative blank control. The α-glucosidase inhibition rate is calculated according to the following formula:

[0067] α-glucosidase inhibition rate (%) = [1- (As-Ase) / (Ac-Ace)] × 100 Formula 2

[0068] In formula 2, As represents the absorbance value of the sample; Ase represents the absorbance value of the sample blank control; Ac represents the absorbance value of the negative control; Ace represents the absorbance value of the negative blank control.

[0069] The chickpea protein peptide solution was diluted to different concentrations and the α-glucosidase inhibition rate was determined. The α-glucosidase inhibition rate was plotted as the vertical axis and the log value of the sample concentration was plotted as the horizontal axis. GraphPad Prism 8.0 was used for nonlinear fitting to calculate the half inhibitory concentration (IC 50 ).

[0070] The α-amylase inhibition rate was determined as follows: chickpea protein peptide was dissolved in deionized water to prepare a sample solution with a protein concentration of 10 mg / mL. A 3,5-dinitrosalicylic acid solution (96 mM, 20 mL) was mixed with a 5.3 M sodium tartrate tetrahydrate solution (8 mL, 2 M sodium hydroxide solution) to prepare the 3,5-dinitrosalicylic acid reagent. 250 μL of the sample solution was mixed with 250 μL of a soluble starch solution (1% w / v, dissolved in 0.1 M sodium phosphate buffer, pH 6.9). The mixture was preheated at 37°C for 10 min, and 250 μL of an α-amylase solution (0.5 U / mL, dissolved in 0.02 M sodium phosphate buffer containing 6 mM NaCl, pH 6.9) was added. The mixture was incubated at 37°C for 10 min. Add 500 μL of 3,5-dinitrosalicylic acid reagent to terminate the reaction. After heating in a boiling water bath for 10 minutes, take 75 μL of the reaction solution and dissolve it in 500 μL of deionized water. Measure the absorbance at 540 nm. Use 0.02 M sodium phosphate buffer containing 6 mM NaCl instead of the α-amylase solution as a sample blank control, and deionized water instead of the sample as a negative control. Use 0.02 M sodium phosphate buffer containing 6 mM NaCl instead of the α-amylase solution and deionized water instead of the sample solution as a negative blank control. Calculate the α-amylase inhibition rate according to the following formula:

[0071] α-Amylase inhibition rate (%) = [1- (As-Ase) / (Ac-Ace)] × 100 Formula 3

[0072] In formula 3, As represents the absorbance value of the sample; Ase represents the absorbance value of the sample blank control; Ac represents the absorbance value of the negative control; Ace represents the absorbance value of the negative blank control.

[0073] The chickpea protein peptide solution was diluted to different concentrations and the α-amylase inhibition rate was determined. The α-amylase inhibition rate was plotted as the vertical axis and the log value of the sample concentration was plotted as the horizontal axis. GraphPad Prism 8.0 was used to perform nonlinear fitting to calculate the half inhibitory concentration (IC 50 ).

[0074] Table 1 DPP-IV, α-glucosidase and α-amylase inhibition rates of chickpea protein hydrolyzed by different proteases

[0075]

[0076]

[0077] In Table 1, CP represents undigested chickpea protein isolate, Pep represents pepsin, Pan represents pancreatin, AoNPI represents neutral protease derived from Aspergillus oryzae 3.042, Aci represents acid protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10012), Neu represents neutral protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10013), Pap represents papain (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10011), Pro represents composite protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10155), Bro represents bromelain (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10010), Fla represents flavor protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10153), and Alc represents alkaline protease (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10154). Different lowercase superscript letters in the same column indicate significant differences between the corresponding data of different enzymatic hydrolysis products (P<0.05).

[0078] According to Table 1, when chickpea protein isolate was enzymatically hydrolyzed in sequence using neutral protease (AoNPI) derived from Aspergillus oryzae 3.042, pepsin (Pep) and pancreatin (Pan), the resulting enzymatic hydrolysates had relatively high inhibition rates on DPP-IV, α-glucosidase and α-amylase, which were 31.26±0.82%, 27.44±0.97% and 41.80±0.74%, respectively.

[0079] Therefore, the enzymatic hydrolysis steps were determined as follows: 1500 U / g of Aspergillus oryzae neutral protease was added according to the weight of chickpea protein isolate, the reaction was carried out at 55°C for 1 hour, and after the reaction, the enzyme was inactivated by heating in a water bath at 100°C for 5 minutes. The pH of the solution was adjusted to 2.0, 2000 U / g of pepsin was added according to the amount of protein, the reaction was carried out at 37°C for 2 hours, and after the reaction, the enzyme was inactivated by heating in a water bath at 100°C for 5 minutes. The pH of the solution was adjusted to 7.0, 2000 U / g of pancreatin was added according to the amount of protein, the reaction was carried out at 37°C for 2 hours, and after the reaction, the enzyme was inactivated by heating in a water bath at 100°C for 5 minutes. After cooling to room temperature, the solution was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected.

[0080] Example 2: Separation of chickpea protein hydrolysate

[0081] 1. Use Sartorius's 10kDa and 3kDa ultrafiltration membrane packages ( 50) Separate 1 mg / mL chickpea protein hydrolysate, collect fractions with molecular weights <3 kDa, 3-10 kDa, and >10 kDa, freeze-dry, and assay for DPP-IV, α-glucosidase, and α-amylase inhibitory activities. Figure 1 As shown in Figure 2, it can be seen that the components with molecular weight <3 kDa have better inhibitory activity against DPP-IV, α-glucosidase and α-amylase, and the corresponding IC 50 They are 8.24 mg / mL, 7.44 mg / mL and 13.23 mg / mL respectively.

[0082] 2. Adoption A rapid protein purification liquid chromatography system was coupled with a Sephadex G-15 column (1000 mm × 12 mm) to separate ultrafiltration fractions with a molecular weight <3 kDa. The sample volume was 2 mL, the eluent was 10 mmol / L phosphate buffer (pH 7.0), the elution rate was 0.8 mL / min, and the UV detection wavelength was 280 nm. The peak patterns of the components are shown in Figure 2. Figure 2 As shown, the individual fractions F1-F7 were collected, freeze-dried and tested for DPP-IV, α-glucosidase and α-amylase inhibitory activities. Figure 3 shown.

[0083] according to Figure 3 It can be seen that the F7 fraction has the highest inhibitory activity against DPP-IV, α-glucosidase and α-amylase, and the corresponding IC 50 They are 0.09 mg / mL, 0.12 mg / mL and 0.06 mg / mL respectively. The F7 component is chickpea protein peptide.

[0084] The peptide composition of the F7 fraction is shown in Table 2, and a total of 526 peptides were identified. Using the AntiT2DMP-Pred tool (https: / / babalab-skku.org / AntiT2DMP-Pred / ), 155 peptides were identified as having hypoglycemic activity. Seven peptides had a confidence level greater than 0.9 for hypoglycemic activity, namely FGL, GLSVFVPLNL, SFGW, SGW, YGM, YGW, and YWM. Fifteen peptides had a confidence level between 0.8 and 0.9 for hypoglycemic activity, namely EDW, ELW, FEGW, FGY, FPW, FSW, GCFSCNSGTG, GFGP, LGW, LLGGF, VGF, VPW, WSFG, WVP, and YGF. Therefore, the above peptides have potential hypoglycemic activity.

[0085] Table 2 Peptide composition of F7 component and its predicted hypoglycemic activity

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] Example 3: Inhibitory effect of chickpea protein peptides on DPP-IV, glycoside hydrolase and glucose transport in Caco-2 cells

[0100] 1. Culture Caco-2 cells in Caco-2 cell culture medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin, i.e., complete culture medium. Maintain the culture medium at 37°C and 5% CO2, and replace the complete culture medium every two days. Discard the culture medium when the cell growth area reaches 70%-80% of the culture dish area (100 mm × 21 mm). Wash the cells twice with 1 mL of phosphate-buffered saline (PBS, pH 7.4, 0.01 M). Add 1 mL of 0.25% trypsin-EDTA solution and digest the cells at 37°C for 2 min. Discard the digestion by adding 5 mL of complete culture medium. Collect the digested cell suspension, centrifuge it (800 rpm, 5 min), discard the culture medium, and add 3 mL of complete culture medium. Use a pipette tip to gently pipette 15-20 times to resuspend the cells. Take 1 mL of the cell suspension and inoculate it into a new culture dish containing 7 mL of complete culture medium. After mixing, culture it at 37°C and 5% CO2.

[0101] When the Caco-2 cells grew to 80%, the cell density was 4 × 10 5The cell suspension of 100 cells / mL was inoculated into the apical side (AP) of a 12-well polycarbonate membrane culture plate (Corning Transwell 3460) with an addition volume of 0.5 mL, and 1.5 mL of complete culture medium was added to the basolateral side (BL). Cultured for 21 days, the medium was changed every other day in the first week until the cells formed a complete membrane, and the medium was changed every day from the second week onwards. The integrity of the cell monolayer membrane was evaluated by measuring the transendothelial electrical resistance (TEER). The TEER value calculation formula is as follows. When the resistance is greater than 400Ω·cm 2 The Caco-2 cell monolayer model was successfully established and can be used for subsequent experiments.

[0102] TEER (Ω·cm 2 ) = (Ω – Ω0) × 1.12 Equation 4

[0103] In formula 4, Ω represents the transmembrane resistance of the cell monolayer; Ω0 represents the blank resistance without cells; 1.12 is the effective membrane area (cm2) on the side of the 12-well Transwell AP. 2 ).

[0104] 2. Cytotoxicity evaluation: The cell density was 1×10 5 A Caco-2 cell suspension of 100 μL cell / mL was inoculated into a 96-well plate, 100 μL cell suspension was added to each well, and the cells were cultured at 5% CO2 and 37°C for 24 hours, and the culture medium was discarded. DMEM culture medium containing only 1% (v / v) penicillin-streptomycin, i.e., an incomplete culture medium, was added, and the cells were cultured at 5% CO2 and 37°C for 4 hours, and the culture medium was discarded. Then, 0.05-0.20 mg / mL chickpea protein peptide solution (dissolved in complete culture medium) was added and cultured for 24 hours, and the culture medium was discarded. Complete culture medium was used to prepare a cell proliferation and toxicity detection reagent (Cell Counting Kit-8, CCK-8) containing 10% (v / v), 100 μL was added to each well, cultured for 1 hour, and the absorbance at 450 nm was measured using an enzyme reader. Complete culture medium was used instead of chickpea protein peptide solution as the control group, and complete culture medium was used instead of cell suspension and chickpea protein peptide solution as the blank group. The calculation formula for cell viability is as follows:

[0105] Cell survival rate (%) = (A s -A b ) / (A c -A b ) × 100% Formula 5

[0106] In formula 5, A sIndicates the absorbance value of the experimental group; A c represents the absorbance value of the control group; A b Represents the absorbance value of the blank group.

[0107] The results are as follows Figure 4 As shown, it can be seen that under the culture conditions of different concentrations of chickpea protein peptide, the survival rate of Caco-2 cells is higher than 89%, indicating that the chickpea protein peptide has no cytotoxicity when the concentration is 0.05-0.20 mg / mL.

[0108] 3. Inhibitory effect of chickpea protein peptides on DPP-IV in Caco-2 cells: using DPPIV-Glo TM The inhibitory activity of chickpea protein peptide on DPP-IV secretion of Caco-2 cells was determined by protease kit. Caco-2 cells were plated at 2×10 5 The cells were inoculated into 12-well plates at a density of 100 cells / well, cultured at 5% CO2 and 37°C for 24 hours, and the culture medium was discarded. 1 mL of incomplete culture medium was added, cultured at 5% CO2 and 37°C for 4 hours, and the culture medium was discarded. Then, 1 mL of chickpea protein peptide solution (0.05-0.20 mg / mL, dissolved in complete culture medium) was added and cultured for 24 hours, and the upper layer of cell culture fluid was collected. 50 μL of cell culture fluid was added to a 96-well plate, and then 50 μL of DPPIV-Glo was added. TM The reagents were shaken and mixed for 30 seconds. The reaction was allowed to react at room temperature for 30 minutes. The relative luminescence signal was measured using a chemiluminescence microplate reader. Complete culture medium was used instead of the chickpea protein peptide solution as a negative control, and 0.05 mg / mL diprotin A (dissolved in complete culture medium) was used instead of the chickpea protein peptide solution as a positive control.

[0109] The results are as follows Figure 5 As shown in the figure, at the concentration of chickpea protein peptide of 0.10 and 0.20 mg / mL, the inhibitory activity of chickpea protein peptide on DPP-IV on Caco-2 cells was manifested by reducing the luminescence signal to 2.84×10 5 RLU and 1.86×10 5 RLU.

[0110] 4. Inhibitory effect of chickpea protein peptide on maltase and sucrase on Caco-2 cells: 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin were added to high-glucose DMEM medium (glucose concentration of 4.5 g / L) to prepare a high-glucose complete medium. 0.5 mL and 1.5 mL of high-glucose complete medium were added to the AP side and BL side of the Caco-2 cell monolayer, respectively, and cultured at 5% CO2 and 37°C for 24 hours. A solution containing 20 mM maltose and 50 mM sucrose (maltose-sucrose, MS) was prepared using a high-glucose complete medium, and then a 0.05-0.20 mg / mL chickpea protein peptide solution was prepared using the MS solution. 800 μL of chickpea protein peptide solution was added to the AP side, and 1.5 mL of high-glucose complete medium was added to the BL side. An MS solution containing 0.05 mg / mL acarbose was used instead of the chickpea protein peptide solution as a positive control, and an MS solution was used as a negative control. After incubation at 5% CO₂ and 37°C for 24 hours, the solutions were collected from the AP and BL sides, and the maltose and sucrose contents in the collected solutions were determined using high-performance liquid chromatography-differential refractive index detector (HPLC-RID). The inhibition rates of maltase and sucrase were calculated as follows:

[0111] Maltase / sucrase inhibition rate (%) = 1 – [(C0 – C' a – C' b ) / (C0 – C a – C b )] × 100% Formula 6

[0112] In formula 6, C0 represents the initial content of maltose / sucrose on the AP side of the negative control; C a Represents the maltose / sucrose content on the AP side after negative control culture; C b Represents the maltose / sucrose content of the BL side after negative control culture; C' a Indicates the content of maltose / sucrose on the AP side of the experimental group after culture; C' b It indicates the maltose / sucrose content on the BL side after culture in the experimental group.

[0113] The results are as follows Figure 6-7 As shown, at a concentration of 0.20 mg / mL, the chickpea protein peptide had an inhibition rate of 42.22% on sucrase and 51.44% on maltase in Caco-2 cells.

[0114] 5. Effect of chickpea protein peptides on glucose transport in Caco-2 cells: 0.5 mL and 1.5 mL of PBS buffer were added to the AP and BL sides of the Caco-2 cell monolayer, respectively. The cells were equilibrated at 5% CO₂ and 37°C for 30 min, and the PBS buffer was discarded. High-glucose (4.5 g / L) and low-glucose DMEM (1 g / L) complete medium were prepared by adding 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin. Subsequently, 0.05-0.20 mg / mL chickpea protein peptide solutions were prepared using high-glucose and low-glucose complete medium, respectively. 0.6 mL of chickpea protein peptide solution was added to the AP side, and 1.2 mL of PBS buffer was added to the BL side. The cells were incubated at 5% CO₂ and 37°C for 2 h. The BL side solution was collected, and the glucose content was determined using a glucose assay kit. PBS buffer was used instead of chickpea protein peptide solution as a blank control, high-glucose or low-glucose complete medium was used instead of chickpea protein peptide solution as a negative control, and 0.05 mg / mL phlorizin (dissolved in high-glucose or low-glucose complete medium) was used instead of chickpea protein peptide solution as a positive control. The formula for calculating the glucose transport inhibition rate is as follows:

[0115] Glucose transport inhibition rate (%) = (C a – C b ) / (C a – C0) × 100% Formula 7

[0116] In formula 7, C a represents the glucose concentration of the negative control; C b represents the glucose concentration of the experimental group; C0 represents the glucose concentration of the blank control.

[0117] The results are as follows Figure 8 As shown, the chickpea protein peptide has an inhibition rate of 50.65%-52.38% on high / low concentration glucose transport in Caco-2 cells at a concentration of 0.2 mg / mL, and the inhibitory effect is dose-dependent with the concentration of the chickpea protein peptide.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing chickpea protein peptide, characterized in that: include: S1. Obtaining chickpea protein isolate, and mixing the chickpea protein isolate with deionized water to prepare a protein suspension; S2, adding neutral protease, pepsin and pancreatin to the protein suspension in sequence to enzymolyze the protein in sequence, and collecting the enzymolysis products after the enzymolysis is completed; the neutral protease is derived from Aspergillus oryzae 3.042; S3, ultrafiltration of the enzymatic hydrolysate using an ultrafiltration membrane with a cut-off of 3 kDa to collect components with a molecular weight less than 3 kDa; S4. Use a Sephadex G-15 chromatography column to perform gel chromatography on the components with a molecular weight less than 3 kDa, the eluent is a phosphate buffer, the concentration of the phosphate buffer is 10 mmol / L, the pH is 7.0, the ultraviolet detection wavelength is 280 nm, the elution rate is 0.8 mL / min, and the components flowing out with an elution time of 160-191 min are collected, and the chickpea protein peptide is obtained after drying.

2. The preparation method according to claim 1, characterized in that Based on each gram of chickpea protein isolate, the added amount of the neutral protease is 1000-2000U.

3. The preparation method according to claim 1, characterized in that Based on each gram of chickpea protein isolate, the added amount of pepsin is 1000-2000U.

4. The preparation method according to claim 1, characterized in that Based on each gram of chickpea protein isolate, the added amount of the pancreatic enzyme is 1000-2000 U.

5. The preparation method according to any one of claims 1 to 4, characterized in that The neutral protease has an enzymatic hydrolysis time of 1-2 hours on the chickpea protein isolate and a pH of 7.0-9.

0.

6. The preparation method according to any one of claims 1 to 4, characterized in that The enzymatic hydrolysis time of the chickpea protein isolate by the pepsin is 1-2 hours, and the pH value is 1.5-3.

0.

7. The preparation method according to any one of claims 1 to 4, characterized in that The enzymatic hydrolysis time of the chickpea protein isolate by the pancreatic enzyme is 1-2 hours, and the pH value is 6.0-8.

0.

8. Chickpea protein peptide prepared according to the preparation method according to any one of claims 1 to 7.

9. The chickpea protein peptide according to claim 8, characterized in that The chickpea protein peptide simultaneously has DPP-IV inhibitory activity, α-glucosidase inhibitory activity and α-amylase inhibitory activity.

10. Use of the chickpea protein peptide according to any one of claims 8 to 9 in the preparation of a product with blood sugar lowering function.

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