Method for extracting bioactive peptide with antioxidant and hypoglycemic effects from xanthoceras sorbifolia bunge meal

Abstract

The invention discloses a method for extracting bioactive peptide with antioxidant and hypoglycemic effects from shiny-leaved yellowhorn meal, and belongs to the technical field of biochemistry. The xanthoceras sorbifolia bunge active peptide with the effects of resisting oxidation and reducing blood sugar is obtained through the operations of high heat, ultrahigh pressure pretreatment, enzymolysis and the like on xanthoceras sorbifolia bunge meal. In-vivo experiments show that the xanthoceras sorbifolia bunge bioactive peptide prepared by the method can reduce the fasting blood-glucose level of diabetic mice, relieve polydipsia symptoms of the mice, gradually increase the weight of the diabetic mice, improve the glucose regulation capability of the diseased mice, relieve liver injury caused by diabetes and improve the diabetes mellitus activity of the diseased mice, so that the xanthoceras sorbifolia bunge bioactive peptide can be used for treating diabetes mellitus. And a good protection effect is achieved on liver cells. Therefore, the bioactive peptide which is extracted from the xanthoceras sorbifolia bunge meal and has the effects of resisting oxidation and reducing blood sugar has a remarkable blood sugar reducing effect, has a wide application prospect in preparation of products for resisting oxidation and reducing blood sugar, and is natural in components and high in safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to a method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal. Background Art

[0002] Xanthoceras sorbifolium Bunge (scientific name: Xanthoceras sorbifolium Bunge) is a deciduous shrub or small tree belonging to the family Sapindaceae and the genus Xanthoceras. Currently, the reported effects of Xanthoceras sorbifolium Bunge active peptides mainly include blood pressure lowering, antioxidant, and acetylcholinesterase inhibitory activities. There is no report on Xanthoceras sorbifolium Bunge active peptides with antioxidant and hypoglycemic effects.

[0003] Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by hyperglycemia caused by various reasons. Its management mainly relies on oral hypoglycemic drugs to control blood sugar levels. Commonly used oral hypoglycemic drugs include α-glucosidase inhibitors, sulfonylureas, biguanides, etc. These drugs have indeed played an important role in lowering blood sugar. However, they are also accompanied by a series of obvious side effects, which not only affect the treatment compliance of patients but also may increase the treatment cost. Therefore, finding natural foods as safe substitutes for preventing / treating T2DM has attracted much attention. In recent years, food-derived bioactive peptides have been regarded as an important strategy for diabetes treatment. Thus, extracting natural bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolium Bunge not only lays a foundation for the development of green, safe, and highly active hypoglycemic products but also helps to promote the high-value utilization of Xanthoceras sorbifolium Bunge meal by-products. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolium Bunge meal.

[0005] In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0006] A method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolium Bunge meal, the steps are as follows:

[0007] (1) According to a mass ratio of 1:10 to 1:30, dissolve defatted Xanthoceras sorbifolium Bunge meal powder in deionized water, stir evenly to obtain a defatted Xanthoceras sorbifolium Bunge powder solution;

[0008] (2) Pretreatment: Place the defatted Xanthoceras sorbifolium Bunge powder solution in a water bath, perform high-temperature heat treatment at 50 - 100 °C for 10 - 50 minutes, and then perform ultra-high pressure pretreatment at 150 - 350 MPa for 150 - 350 s;

[0009] (3) Enzymatic hydrolysis: Add protease to the pretreated solution, mix evenly, and perform enzymatic hydrolysis at 40 - 60°C for 1 - 5 hours with a pH of 6 - 10; centrifuge the enzymatically hydrolyzed solution and take the supernatant to obtain the product.

[0010] Based on the above - mentioned scheme, the protease is at least one of alkaline protease, compound protease, and flavor protease; the addition amount of the protease is 3000 - 5000 U / g.

[0011] Based on the above - mentioned scheme, the protease is alkaline protease, compound protease, and flavor protease; the addition amount of each protease is 4500 U / g.

[0012] Based on the above - mentioned scheme, in step (1), the mass ratio of defatted Xanthoceras sorbifolia Bunge meal powder to deionized water is 1:10.

[0013] Based on the above - mentioned scheme, in step (2), the high - temperature treatment temperature is 80°C and the treatment time is 30 min.

[0014] Based on the above - mentioned scheme, in step (2), the ultra - high - pressure pretreatment pressure is 200 MPa and the pretreatment time is 350 s.

[0015] Based on the above - mentioned scheme, in step (3), the enzymatic hydrolysis time is 4 hours and the temperature is 50°C.

[0016] Based on the above - mentioned scheme, the method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal further includes the following steps: Subject the supernatant obtained in step (3) to molecular weight fractionation through an ultrafiltration centrifuge tube, and collect the fraction with a molecular weight of 500 Da - 3 kDa.

[0017] Application of the bioactive peptide with antioxidant and hypoglycemic effects prepared by the above - mentioned method in the preparation of products with antioxidant and hypoglycemic effects, where the products are food, drugs, or health products.

[0018] Based on the above - mentioned scheme, the antioxidant effects include DPPH scavenging activity, hydroxyl radical scavenging activity, and ferric ion reducing ability; the hypoglycemic effects include the activities of inhibiting α - glucosidase and inhibiting α - amylase.

[0019] Advantages of the technical solution of the present invention

[0020] The present invention discloses a method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolium Bunge meal. Through operations such as high-temperature and ultra-high-pressure pretreatment and enzymatic hydrolysis of Xanthoceras sorbifolium Bunge meal, Xanthoceras sorbifolium Bunge peptides with high hypoglycemic activity are obtained. In vitro experiments show that the Xanthoceras sorbifolium Bunge polypeptides prepared by the present invention simultaneously possess high antioxidant activity, α-glucosidase inhibitory activity, and α-amylase inhibitory activity; in vivo experiments show that the Xanthoceras sorbifolium Bunge bioactive peptides prepared by the method of the present invention can reduce the fasting blood glucose level of diabetic mice, relieve the polyphagia symptom of mice, gradually increase the body weight of diabetic mice, improve the glucose regulation ability of diseased mice, and can relieve the liver damage caused by diabetes, having a good protective effect on liver cells. Moreover, it also has a regulatory effect on the intestinal flora disorder caused by diabetes in mice, and indirectly affects the regulation of blood glucose by intestinal microorganisms through the regulation of intestinal flora. Therefore, the bioactive peptides with antioxidant and hypoglycemic effects extracted from Xanthoceras sorbifolium Bunge meal by the present invention have a significant hypoglycemic effect, have broad application prospects in the preparation of hypoglycemic products, and have natural components and high safety. In addition, the extraction of bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolium Bunge meal by the present invention is also conducive to promoting the high-value utilization of Xanthoceras sorbifolium Bunge meal by-products and expanding the application fields of Xanthoceras sorbifolium Bunge. Description of the Drawings

[0021] Figure 1 Effects of different proteases on the enzymatic hydrolysis effect of Xanthoceras sorbifolium Bunge and the activity of products (wherein, Alc: alkaline protease; Pro: compound protease; Fla: flavor protease; different letters indicate significant differences, p < 0.05);

[0022] Figure 2 Effect of different high-temperature treatment temperatures on the α-glucosidase inhibitory activity of Xanthoceras sorbifolium Bunge peptides;

[0023] Figure 3 Effect of different high-temperature treatment times on the α-glucosidase inhibitory activity of Xanthoceras sorbifolium Bunge peptides;

[0024] Figure 4 Effect of different ultra-high-pressure pressures on the α-glucosidase inhibitory activity of Xanthoceras sorbifolium Bunge peptides;

[0025] Figure 5 Effect of different ultra-high-pressure times on the α-glucosidase inhibitory activity of Xanthoceras sorbifolium Bunge peptides;

[0026] Figure 6 Effect of different solid-liquid ratios on the α-glucosidase inhibitory activity of Xanthoceras sorbifolium Bunge peptides;

[0027] Figure 7 Effect of different enzyme addition amounts on the α-glucosidase inhibitory activity of Xanthoceras sorbifolium Bunge peptides;

[0028] Figure 8Effect of different enzymatic hydrolysis temperatures on the α-glucosidase inhibitory activity of Xanthoceras sorbifolia peptide;

[0029] Figure 9 Effect of different enzymatic hydrolysis times on the α-glucosidase inhibitory activity of Xanthoceras sorbifolia peptide;

[0030] Figure 10 Effect of different enzymatic hydrolysis pH values on the α-glucosidase inhibitory activity of Xanthoceras sorbifolia peptide;

[0031] Figure 11 Changes in blood glucose levels of mice weekly within 6 weeks of dietary intervention ("" indicates a significant difference between the sample group and the control group, p < 0.01; "***" indicates a statistically significant difference between the sample group and the model group, p < 0.01);

[0032] Figure 12 Effect of changes in water intake of mice during dietary intervention ("" indicates a significant difference between the sample group and the control group, p < 0.01; "***" indicates a statistically significant difference between the sample group and the model group, p < 0.01; "ns" indicates no statistical difference);

[0033] Figure 13 Effect of changes in body weight of mice during dietary intervention ("" indicates a significant difference between the sample group and the control group, p < 0.01; "**" indicates a statistically significant difference between the sample group and the model group, p < 0.05);

[0034] Figure 14 Results of the oral glucose tolerance test (OGTT) in mice ("" indicates a significant difference between the sample group and the control group, p < 0.01; "***" indicates a statistically significant difference between the sample group and the model group, p < 0.01);

[0035] Figure 15 Area under the curve (AUC) of the time-blood glucose curve ("" indicates a significant difference between the sample group and the control group, p < 0.01; "***" indicates a statistically significant difference between the sample group and the model group, p < 0.01);

[0036] Figure 16 Results of the effect of Xanthoceras sorbifolia peptide on the levels of TG and TC in mice (the left figure is for TG and the right figure is for TC; "" indicates a significant difference between the sample group and the control group, p < 0.01; "##" indicates a significant difference between the sample group and the control group, p < 0.05; "***" indicates a statistically significant difference between the sample group and the model group, p < 0.01; "**" indicates a statistically significant difference between the sample group and the model group, p < 0.05);

[0037] Figure 17Results of the effects of Xanthoceras sorbifolia peptide on the levels of HDL-C and LDL-C in mice (where the left figure is HDL-C and the right figure is LDL-C; "" indicates a significant difference between the sample group and the control group, p < 0.01; "##" indicates a significant difference between the sample group and the control group, p < 0.05; "***" indicates a statistically significant difference between the sample group and the model group, p < 0.01; "**" indicates a statistically significant difference between the sample group and the model group, p < 0.05);

[0038] Figure 18 Results of the effects of Xanthoceras sorbifolia peptide on the hepatic glycogen content and GSH in mice (where the left figure is the hepatic glycogen content and the right figure is GSH; "" indicates a significant difference between the sample group and the control group, p < 0.01; "***" indicates a statistically significant difference between the sample group and the model group, p < 0.01; "**" indicates a statistically significant difference between the sample group and the model group, p < 0.05);

[0039] Figure 19 Results of the effects of Xanthoceras sorbifolia peptide on AST and ALT in mice (where the left figure is AST and the right figure is ALT; "" indicates a significant difference between the sample group and the control group, p < 0.01; "***" indicates a statistically significant difference between the sample group and the model group, p < 0.01; "**" indicates a statistically significant difference between the sample group and the model group, p < 0.05);

[0040] Figure 20 Results of HE staining of mouse liver tissues (magnification ×200, and the red box indicates the lesion area);

[0041] Figure 21 Results of oil red staining of mouse kidney tissues (magnification ×200, and the black arrow indicates the lesion area);

[0042] Figure 22 Statistical results of the area of oil red staining of mouse liver ("##" indicates a significant difference between the sample group and the control group, p < 0.05; "**" indicates a statistically significant difference between the sample group and the model group, p < 0.05);

[0043] Figure 23 Rank-Abundance curves based on OTU abundances;

[0044] Figure 24 OTU dilution curves;

[0045] Figure 25 Venn diagrams of intestinal flora;

[0046] Figure 26 α-diversity indices of intestinal flora Ace, Chao 1, and Shannon;

[0047] Figure 27 PCoA analysis of the intestinal flora of mice;

[0048] Figure 28 NMDS analysis of the intestinal flora of mice;

[0049] Figure 29 Changes in the intestinal flora of mice at the phylum level;

[0050] Figure 30 Relative abundances of Bacteroidetes, Firmicutes, and Proteobacteria in each group of flora;

[0051] Figure 31 Ratio of Firmicutes / Bacteroidetes;

[0052] Figure 32 Changes in the intestinal flora of mice at the genus level; Detailed implementation mode

[0053] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. The present invention will be described in further detail below in conjunction with specific examples and with reference to the data. The following examples are only for illustrative purposes of the present invention and do not limit the scope of the present invention in any way.

[0054] The experimental methods in the following examples are all conventional methods, unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The test materials, reagents, drugs, etc. used in the following examples can be obtained through general channels, unless otherwise specified.

[0055] 1. Hydrolysis degree measurement method: Use the ninhydrin colorimetric method for measurement. Specifically:

[0056] Mix 2.0 mL of a properly diluted protein hydrolysate solution with 1.0 mL of ninhydrin solution in a centrifuge tube. Next, place the centrifuge tube in an 80 °C water bath for incubation for 30 minutes and cool to room temperature. Subsequently, add 5 mL of distilled water, let stand for 10 min, and measure the absorbance of the solution at 570 nm. Use a glycine standard solution (0, 8, 16, 24, 32, 40 μg / mL) instead of the protein hydrolysate solution as the working curve. Calculate the degree of hydrolysis DH according to the following formula.

[0057]

[0058] DH is defined as the percentage of cleaved peptide bonds, where ht is the total number of peptide bonds per protein equivalent and h is the number of hydrolyzed bonds.

[0059] 2. Method for measuring α-glucosidase inhibition rate, steps are as follows:

[0060] Mix 200 μL of protein solution and 200 μL of 0.1 U / mL -1 α-glucosidase (0.2 M phosphate buffer, pH 6.8) at 37 °C for 15 min, then add 200 μL of p-nitrophenyl-α-D-glucoside (2.5 mmol / L -1 ), make up to 2 mL with PBS, and mix at 37 °C for 30 min. Add 2 mL of 0.2 mol / L -1 Na2CO3 to terminate the reaction. Measure the absorbance (OD C ) at 405 nm using a spectrophotometer to calculate α-glucosidase activity. Another blank group (OD A ), control group (OD B ), and sample background group (OD D ) are set in the experiment. Calculate the α-glucosidase inhibition rate of the protein solution to be measured according to the following formula:

[0061]

[0062] 3. Method for measuring α-amylase inhibition rate, steps are as follows:

[0063] Mix 100 μL of protein solution with 100 μL of α-amylase (0.5 U / mL -1 phosphate buffer, pH 6.8) at 37 °C for 10 min. Then add 125 μL of 1% gelatinized starch (0.2 M phosphate buffer, pH 6.8), incubate at 37 °C for 10 min. Add 150 μL of 3,5-dinitrosalicylic acid (DNS) reagent to terminate the reaction after 5 min. Measure the absorbance OD C at 540 nm using a spectrophotometer. Another blank group (OD A ), control group (OD B ), and sample background group (OD D ) are set in the experiment. Calculate the α-amylase activity according to the following formula.

[0064]

[0065] 4. DPPH scavenging activity

[0066] Measure the DPPH scavenging activity of the peptide, and use glutathione (GSH) as the positive control. Mix 2 mL of Xanthoceras sorbifolia Bunge peptide sample and 2.5 mL of DPPH-absolute ethanol solution (100 μM) evenly, let stand in the dark for 30 min, and measure the absorbance value at 517 nm. Another blank group (OD A ), control group (OD B) and the sample background group (OD D ). Calculate the scavenging rate of the sample to be measured on DPPH free radicals according to the following formula:

[0067]

[0068] 5. Hydroxyl radical scavenging rate

[0069] Measure the hydroxyl radical scavenging activity of the peptide, and use glutathione (GSH) as a positive control. Add 1 mL of FeSO4 solution (6 mM), 1 mL of salicylic acid solution (6 mM), 1 mL of Xanthoceras sorbifolia Bunge peptide (10 mg / mL), and 1 mL of H2O2 solution (6 mM) to a colorimetric test tube. Shake them well and place them at 37 °C for 30 minutes. Detect its absorbance OD C . Another blank group (OD A ), control group (OD B ), and sample background group (OD D ) are set up in the experiment. Calculate the hydroxyl radical scavenging ability according to the following formula:

[0070]

[0071] 6. Ferric ion reducing ability

[0072] Measure the ferric ion reducing activity of the peptide, and use glutathione (GSH) as a positive control. Add 1 mL of phosphate buffer solution (0.2 mol / L with pH = 6.6), then prepare 1 mL of K3[Fe(CN)6] (1%) and Xanthoceras sorbifolia Bunge peptide (10 mg / mL) and add them to a colorimetric tube. After mixing evenly, react the solution in a 50 °C water bath for 20 min, and finally add 1 mL of TCA (10%) to terminate the reaction. Centrifuge the mixture at 5000×g for 10 minutes. After standing for 10 minutes, take 2.5 mL of the supernatant, 2.5 mL of distilled water, and 1.2 mL of FeCl3 (0.1%) and measure the absorbance value OD of the sample at 700 nm C . Another blank group (OD A ), control group (OD B ), and sample background group (OD D ) are set up in the experiment. Calculate the ferric ion reducing ability according to the following formula:

[0073]

[0074] Effects of different proteases on the enzymatic hydrolysis effect of Xanthoceras sorbifolia Bunge and the activity of the products

[0075] (1) Select fresh defatted Xanthoceras sorbifolia Bunge meal. After cleaning, drying, and pulverizing, dissolve it in deionized water according to a mass ratio of 1:10 to obtain a defatted Xanthoceras sorbifolia Bunge powder solution.

[0076] (2) Pretreatment: Place the defatted Xanthoceras sorbifolium Bunge fruit powder solution in a water bath, perform high-temperature heat treatment at 70 °C for 20 minutes, and then perform ultra-high pressure pretreatment at 200 MPa for 300 s. The pretreated solution shows a more uniform texture, which is beneficial to the subsequent enzymatic hydrolysis process.

[0077] (3) Enzymatic hydrolysis: Add alkaline protease, compound protease, and flavor protease to the defatted Xanthoceras sorbifolium Bunge fruit powder solution in single and compound forms respectively. After mixing evenly, carry out enzymatic hydrolysis at 50 °C for 4 hours, pH 6.8, and the addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolium Bunge fruit powder is 4000 U).

[0078] (4) After enzymatic hydrolysis is completed, centrifuge, collect the supernatant, and measure the degree of hydrolysis, α-glucosidase inhibition rate, and α-amylase inhibition rate. The results are as Figure 1 shown. The best activity is shown when the three enzymes are used in combination. Its α-glucosidase inhibition rate is 58%, the α-amylase inhibition rate is 67%, and the degree of hydrolysis reaches 55%.

[0079] Effect of different high-temperature treatment temperatures on the α-glucosidase inhibitory activity of Xanthoceras sorbifolium Bunge peptides

[0080] (1) Select fresh defatted Xanthoceras sorbifolium Bunge meal, after cleaning, drying, and pulverizing, dissolve it in deionized water according to a mass ratio of 1:10 to obtain a defatted Xanthoceras sorbifolium Bunge fruit powder solution.

[0081] (2) Pretreatment: Place the defatted Xanthoceras sorbifolium Bunge fruit powder solution in a water bath and perform high-temperature heat treatment at different temperatures (50 °C, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C) for 20 minutes, and then perform ultra-high pressure pretreatment at 200 MPa for 300 s.

[0082] (3) Enzymatic hydrolysis: Add protease to the pretreated solution, mix evenly, and carry out enzymatic hydrolysis at 50 °C for 4 hours, pH 6.8. The protease is composed of a compound of alkaline protease, compound protease, and flavor protease. Among them, the addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolium Bunge fruit powder is 4000 U).

[0083] (4) After enzymatic hydrolysis is completed, centrifuge the enzymatic hydrolysis solution at a rotation speed of 8500 rpm for 15 minutes. After centrifugation, separate the supernatant. Measure the α-glucosidase inhibition rate activity of the supernatant. The results are as Figure 2 shown. The α-glucosidase inhibition rate activity of the supernatant first increases and then decreases with the increase of temperature, and 80 °C is the optimal heat treatment temperature.

[0084] Effect of different high-temperature treatment times on the α-glucosidase inhibitory activity of Xanthoceras sorbifolium Bunge peptides

[0085] (1) Select fresh defatted Xanthoceras sorbifolium Bunge meal. After washing, drying, and pulverizing, dissolve it in deionized water at a mass ratio of 1:10 to obtain a defatted Xanthoceras sorbifolium Bunge powder solution.

[0086] (2) Pretreatment: Place the defatted Xanthoceras sorbifolium Bunge powder solution in a water bath and conduct high-temperature heat treatment at 70 °C for different times (10 min, 20 min, 30 min, 40 min, 50 min), and then conduct ultra-high pressure pretreatment at 200 MPa for 300 s.

[0087] (3) Enzymolysis: Add protease to the pretreated solution, mix evenly, and conduct enzymolysis at 50 °C for 4 hours, with a pH of 6.8. The protease is composed of a compound of alkaline protease, compound protease, and flavor protease. Among them, the addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolium Bunge powder is 4000 U).

[0088] (4) After the enzymolysis is completed, centrifuge the enzymolyzed solution at a speed of 8500 rpm for 15 minutes. After centrifugation, separate the supernatant. Measure the α-glucosidase inhibitory rate activity of the supernatant. The results are as Figure 3 shown. The α-glucosidase inhibitory rate activity of the supernatant first increases and then decreases with the increase of the heat treatment time, and 30 min is the optimal heat treatment time.

[0089] Effect of different ultra-high pressure pressures on the α-glucosidase inhibitory activity of Xanthoceras sorbifolium Bunge peptides

[0090] (1) Select fresh defatted Xanthoceras sorbifolium Bunge meal. After washing, drying, and pulverizing, dissolve it in deionized water at a mass ratio of 1:10 to obtain a defatted Xanthoceras sorbifolium Bunge powder solution.

[0091] (2) Pretreatment: Place the defatted Xanthoceras sorbifolium Bunge powder solution in a water bath and conduct high-temperature heat treatment at 70 °C for 20 min, and then conduct ultra-high pressure pretreatment at different pressures (150 MPa, 200 MPa, 250 MPa, 300 MPa, 350 MPa) for 300 s.

[0092] (3) Enzymolysis: Add protease to the pretreated solution, mix evenly, and conduct enzymolysis at 50 °C for 4 hours, with a pH of 6.8. The protease is composed of a compound of alkaline protease, compound protease, and flavor protease. Among them, the addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolium Bunge powder is 4000 U).

[0093] (4) After the enzymatic hydrolysis is completed, the enzymatically hydrolyzed solution is centrifuged at a speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant is separated. The α-glucosidase inhibitory rate activity of the supernatant is measured, and the results are as Figure 4 shown. The α-glucosidase inhibitory rate activity of the supernatant first increases and then decreases with the increase of the ultra-high pressure, and 200 MPa is the optimal ultra-high pressure.

[0094] Effect of Different Ultra-High Pressure Times on α-Glucosidase Inhibitory Activity of Xanthoceras Sorbifolia Bunge Peptide

[0095] (1) Fresh defatted Xanthoceras sorbifolia Bunge meal is selected. After washing, drying, and pulverizing, it is dissolved in deionized water according to a mass ratio of 1:10 to prepare a defatted Xanthoceras sorbifolia Bunge powder solution.

[0096] (2) Pretreatment: The defatted Xanthoceras sorbifolia Bunge powder solution is placed in a water bath and subjected to high-temperature heat treatment at 70 °C for 20 min, and then subjected to ultra-high pressure pretreatment at 200 MPa for different times (150 s, 200 s, 250 s, 300 s, 350 s).

[0097] (3) Enzymatic hydrolysis: Protease is added to the pretreated solution, mixed evenly, and enzymatically hydrolyzed at 50 °C for 4 hours, pH 6.8. The protease is a compound of alkaline protease, compound protease, and flavor protease. Among them, the addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolia Bunge powder is 4000 U).

[0098] (4) After the enzymatic hydrolysis is completed, the enzymatically hydrolyzed solution is centrifuged at a speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant is separated. The α-glucosidase inhibitory rate activity of the supernatant is measured, and the results are as Figure 5 shown. The α-glucosidase inhibitory rate activity of the supernatant increases with the increase of the ultra-high pressure treatment time, and 350 s is the optimal ultra-high pressure treatment time.

[0099] Effect of Different Solid-Liquid Ratios on α-Glucosidase Inhibitory Activity of Xanthoceras Sorbifolia Bunge Peptide

[0100] (1) Fresh defatted Xanthoceras sorbifolia Bunge meal is selected. After washing, drying, and pulverizing, deionized water is added according to different solid-liquid ratios (mass ratios of 1:10, 1:15, 1:20, 1:25, 1:30) to prepare a defatted Xanthoceras sorbifolia Bunge powder solution.

[0101] (2) Pretreatment: The defatted Xanthoceras sorbifolia Bunge powder solution is placed in a water bath and subjected to high-temperature heat treatment at 80 °C for 30 min, and then subjected to ultra-high pressure pretreatment at 200 MPa for 350 s.

[0102] (3) Enzymatic hydrolysis: In the pretreated solution, protease was added and mixed evenly. The hydrolysis was carried out at 50 °C for 4 hours at pH 6.8. The protease is a compound of alkaline protease, compound protease and flavor protease. The addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolia Bunge powder is 4000 U).

[0103] (4) After the enzymatic hydrolysis was completed, the hydrolyzed solution was centrifuged at a speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant was separated. The α-glucosidase inhibitory rate activity of the supernatant was measured, and the results are as Figure 6 shown. The α-glucosidase inhibitory rate activity of the supernatant decreased with the increase of the solid-liquid ratio, and 1:10 was the optimal solid-liquid ratio.

[0104] Effect of Different Enzyme Dosages on the α-Glucosidase Inhibitory Activity of Xanthoceras sorbifolia Bunge Peptide

[0105] (1) Fresh defatted Xanthoceras sorbifolia Bunge meal was selected. After washing, drying and pulverizing, deionized water was added according to the mass ratio of 1:10 to prepare a defatted Xanthoceras sorbifolia Bunge powder solution.

[0106] (2) Pretreatment: The defatted Xanthoceras sorbifolia Bunge powder solution was placed in a water bath and subjected to high-temperature heat treatment at 80 °C for 30 min, and then subjected to ultra-high pressure pretreatment at 200 MPa for 350 s.

[0107] (3) Enzymatic hydrolysis: In the pretreated solution, protease was added at different addition amounts (3000 U / g, 3500 U / g, 4000 U / g, 4500 U / g, 5000 U / g), mixed evenly, and the hydrolysis was carried out at 50 °C for 4 hours at pH 6.8. The protease is a compound of alkaline protease, compound protease and flavor protease. The addition amount of each protease is the same. Taking the protease addition amount of 3000 U / g as an example, "3000 U / g" means that "the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolia Bunge powder is 3000 U".

[0108] (4) After the enzymatic hydrolysis was completed, the hydrolyzed solution was centrifuged at a speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant was separated. The α-glucosidase inhibitory rate activity of the supernatant was measured, and the results are as Figure 7 shown. The α-glucosidase inhibitory rate activity of the supernatant first increased and then decreased with the increase of the enzyme dosage, and 4500 U / g was the optimal enzyme dosage.

[0109] Effect of Different Enzymatic Hydrolysis Temperatures on the α-Glucosidase Inhibitory Activity of Xanthoceras sorbifolia Bunge Peptide

[0110] (1) Select fresh defatted Xanthoceras sorbifolia Bunge meal. After washing, drying, and pulverizing, add deionized water according to a mass ratio of 1:10 to obtain a defatted Xanthoceras sorbifolia Bunge powder solution.

[0111] (2) Pretreatment: Place the defatted Xanthoceras sorbifolia Bunge powder solution in a water bath and perform high-temperature heat treatment at 80 °C for 30 min, followed by ultra-high pressure pretreatment at 200 MPa for 350 s.

[0112] (3) Enzymatic hydrolysis: Add protease to the pretreated solution, mix evenly, and perform enzymatic hydrolysis at different temperatures (40 °C, 45 °C, 50 °C, 55 °C, 60 °C) for 4 hours at pH 6.8. The protease is a compound of alkaline protease, compound protease, and flavor protease. Among them, the addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolia Bunge powder is 4000 U).

[0113] (4) After enzymatic hydrolysis is completed, centrifuge the enzymatically hydrolyzed solution at a speed of 8500 rpm for 15 minutes. After centrifugation, separate the supernatant. Measure the α-glucosidase inhibitory rate activity of the supernatant. The results are as Figure 8 shown. The α-glucosidase inhibitory rate activity of the supernatant first increases and then decreases with the increase of the enzymatic hydrolysis temperature, and 50 °C is the optimal enzymatic hydrolysis temperature.

[0114] Effect of different enzymatic hydrolysis times on the α-glucosidase inhibitory activity of Xanthoceras sorbifolia Bunge peptide

[0115] (1) Select fresh defatted Xanthoceras sorbifolia Bunge meal. After washing, drying, and pulverizing, add deionized water according to a mass ratio of 1:10 to obtain a defatted Xanthoceras sorbifolia Bunge powder solution.

[0116] (2) Pretreatment: Place the defatted Xanthoceras sorbifolia Bunge powder solution in a water bath and perform high-temperature heat treatment at 80 °C for 30 min, followed by ultra-high pressure pretreatment at 200 MPa for 350 s.

[0117] (3) Enzymatic hydrolysis: Add protease to the pretreated solution, mix evenly, and perform enzymatic hydrolysis at 50 °C for different times (1 h, 2 h, 3 h, 4 h, 5 h) at pH 6.8. The protease is a compound of alkaline protease, compound protease, and flavor protease. Among them, the addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolia Bunge powder is 4000 U).

[0118] (4) After enzymatic hydrolysis is completed, centrifuge the enzymatically hydrolyzed solution at a speed of 8500 rpm for 15 minutes. After centrifugation, separate the supernatant. Measure the α-glucosidase inhibitory rate activity of the supernatant. The results are as Figure 9As shown, the α-glucosidase inhibitory rate activity of the supernatant first increases and then decreases with the increase of enzymolysis time, and 4 h is the optimal enzymolysis time.

[0119] Effect of Different Enzymolysis pH on α-Glucosidase Inhibitory Activity of Xanthoceras sorbifolia Bunge Peptide

[0120] (1) Select fresh defatted Xanthoceras sorbifolia Bunge meal. After washing, drying and pulverizing, deionized water is added according to the mass ratio of 1:10 to prepare a defatted Xanthoceras sorbifolia Bunge powder solution.

[0121] (2) Pretreatment: Place the defatted Xanthoceras sorbifolia Bunge powder solution in a water bath and perform high-temperature heat treatment at 80 °C for 30 min, and then perform ultra-high pressure pretreatment at 200 MPa for 350 s.

[0122] (3) Enzymolysis: Add protease to the pretreated solution, mix evenly, and enzymolyze at 50 °C for 4 h, and the pH is set to 6, 7, 8, 9, and 10 respectively. The protease is a compound of alkaline protease, compound protease and flavor protease. Among them, the addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of defatted Xanthoceras sorbifolia Bunge powder substrate is 4000 U).

[0123] (4) After the enzymolysis is completed, centrifuge the enzymolyzed solution at a speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant is separated. Measure the α-glucosidase inhibitory rate activity of the supernatant, and the results are as Figure 10 shown. The α-glucosidase inhibitory rate activity of the supernatant first increases and then decreases with the increase of pH, and pH 7 is the optimal enzymolysis pH.

[0124] Example 1

[0125] A method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal, the steps are as follows:

[0126] (1) Select fresh defatted Xanthoceras sorbifolia Bunge meal. After washing, drying and pulverizing, defatted Xanthoceras sorbifolia Bunge meal powder with uniform particle size is obtained. According to the mass ratio of 1:10, dissolve the defatted Xanthoceras sorbifolia Bunge meal powder in deionized water and stir evenly to prepare a defatted Xanthoceras sorbifolia Bunge powder solution.

[0127] (2) Pretreatment: Place the defatted Xanthoceras sorbifolia Bunge powder solution in a water bath and perform high-temperature heat treatment at 80 °C for 30 minutes, and then perform ultra-high pressure pretreatment at 200 MPa for 350 s. The pretreated solution shows a more uniform texture, which is beneficial to the subsequent enzymolysis process.

[0128] (3) Enzymolysis: Add protease to the pretreated solution, mix evenly, and carry out enzymolysis at 52 °C for 3.5 hours with a pH of 7. The protease is a compound of alkaline protease, compound protease and flavor protease, and the addition amount of each protease is 4500 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolia Bunge fruit powder is 4500 U). During the enzymolysis process, it was observed that the solution gradually became clear, indicating that the protein was effectively decomposed into peptide segments.

[0129] Centrifuge the enzymolyzed solution at a rotation speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant and precipitate are separated. The supernatant is light yellow, highly transparent, and contains a large amount of peptide segments. It will be detected that the α-glucosidase inhibition rate of this supernatant is 41.26%; the α-amylase inhibition rate is 52.89%.

[0130] Example 2

[0131] A method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal, the steps are as follows:

[0132] (1) Select fresh defatted Xanthoceras sorbifolia Bunge meal, wash, dry and crush it to obtain defatted Xanthoceras sorbifolia Bunge fruit powder with uniform particle size. According to the mass ratio of 1:10, dissolve the defatted Xanthoceras sorbifolia Bunge fruit powder in deionized water, stir evenly to prepare a defatted Xanthoceras sorbifolia Bunge fruit powder solution.

[0133] (2) Pretreatment: Place the defatted Xanthoceras sorbifolia Bunge fruit powder solution in a water bath, carry out high-temperature heat treatment at 80 °C for 30 minutes, and then carry out ultra-high pressure pretreatment at 200 MPa for 350 s. The pretreated solution shows a more uniform texture, which is beneficial to the subsequent enzymolysis process.

[0134] (3) Enzymolysis: Add protease to the pretreated solution, mix evenly, and carry out enzymolysis at 50 °C for 4 hours with a pH of 7. The protease is a compound of alkaline protease, compound protease and flavor protease, and the addition amount of each protease is 4500 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolia Bunge fruit powder is 4500 U). During the enzymolysis process, it was observed that the solution gradually became clear, indicating that the protein was effectively decomposed into peptide segments.

[0135] Centrifuge the enzymolyzed solution at a rotation speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant and precipitate are separated. The supernatant is light yellow, highly transparent, and contains a large amount of peptide segments. It will be detected that the α-glucosidase inhibition rate of this supernatant is 77.35%, and the α-amylase inhibition rate is 81.58%.

[0136] Example 3

[0137] A method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolium Bunge meal, the steps are as follows:

[0138] (1) Select fresh defatted Xanthoceras sorbifolium Bunge meal, wash, dry and pulverize it to obtain defatted Xanthoceras sorbifolium Bunge meal powder with uniform particle size. According to the mass ratio of 1:10, dissolve the defatted Xanthoceras sorbifolium Bunge meal powder in deionized water, stir evenly to prepare a defatted Xanthoceras sorbifolium Bunge meal powder solution.

[0139] (2) Pretreatment: Place the defatted Xanthoceras sorbifolium Bunge meal powder solution in a water bath, perform high-temperature heat treatment at 80 °C for 30 minutes, and then perform ultra-high pressure pretreatment at 180 MPa for 350 s. The pretreated solution shows a more uniform texture, which is beneficial to the subsequent enzymatic hydrolysis process.

[0140] (3) Enzymatic hydrolysis: Add protease to the pretreated solution, mix evenly, and perform enzymatic hydrolysis at 50 °C for 3.5 hours, with a pH of 7. The protease is a compound of alkaline protease, compound protease and flavor protease, and the addition amount of each protease is 4500 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolium Bunge meal powder is 4500 U). During the enzymatic hydrolysis process, it is observed that the solution gradually becomes clear, indicating that the protein is effectively decomposed into peptide segments.

[0141] Centrifuge the enzymatically hydrolyzed solution at a speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant and precipitate are separated. The supernatant is light yellow, has high transparency, and contains a large amount of peptide segments. It will be detected that the α-glucosidase inhibition rate of this supernatant is 60.01%; the α-amylase inhibition rate is 73.48%.

[0142] Example 4

[0143] A method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolium Bunge meal, the steps are as follows:

[0144] (1) Select fresh defatted Xanthoceras sorbifolium Bunge meal, wash, dry and pulverize it to obtain defatted Xanthoceras sorbifolium Bunge meal powder with uniform particle size. According to the mass ratio of 1:10, dissolve the defatted Xanthoceras sorbifolium Bunge meal powder in deionized water, stir evenly to prepare a defatted Xanthoceras sorbifolium Bunge meal powder solution.

[0145] (2) Pretreatment: Place the defatted Xanthoceras sorbifolium Bunge meal powder solution in a water bath, perform high-temperature heat treatment at 80 °C for 30 minutes, and then perform ultra-high pressure pretreatment at 200 MPa for 350 s. The pretreated solution shows a more uniform texture, which is beneficial to the subsequent enzymatic hydrolysis process.

[0146] (3) Enzymatic hydrolysis: In the pretreated solution, protease was added and mixed evenly. The enzymatic hydrolysis was carried out at 50 °C for 4 hours with a pH of 7. The protease is a compound of alkaline protease, compound protease and flavor protease, and the addition amount of each protease is 5000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolia Bunge powder is 5000 U). During the enzymatic hydrolysis process, it was observed that the solution gradually became clear, indicating that the protein was effectively decomposed into peptide segments.

[0147] The enzymatically hydrolyzed solution was centrifuged at a speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant and the precipitate were separated. The supernatant was light yellow in color, highly transparent, and contained a large amount of peptide segments. It will be detected that the α-glucosidase inhibition rate of this supernatant was 56.82%; the α-amylase inhibition rate was 62.51%.

[0148] Example 5

[0149] A method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal, the steps are as follows:

[0150] (1) Select fresh defatted Xanthoceras sorbifolia Bunge meal, wash, dry and crush it to obtain defatted Xanthoceras sorbifolia Bunge powder with uniform particle size. According to a mass ratio of 1:10, the defatted Xanthoceras sorbifolia Bunge powder was dissolved in deionized water and stirred evenly to prepare a defatted Xanthoceras sorbifolia Bunge powder solution.

[0151] (2) Pretreatment: The defatted Xanthoceras sorbifolia Bunge powder solution was placed in a water bath and subjected to high-temperature heat treatment at 80 °C for 30 minutes, and then subjected to ultra-high pressure pretreatment at 220 MPa for 350 s. The pretreated solution showed a more uniform texture, which was beneficial to the subsequent enzymatic hydrolysis process.

[0152] (3) Enzymatic hydrolysis: In the pretreated solution, protease was added and mixed evenly. The enzymatic hydrolysis was carried out at 50 °C for 4 hours with a pH of 7. The protease is a compound of alkaline protease, compound protease and flavor protease, and the addition amount of each protease is 4000 U / g (that is, the addition amount of each protease per gram of the substrate defatted Xanthoceras sorbifolia Bunge powder is 4000 U). During the enzymatic hydrolysis process, it was observed that the solution gradually became clear, indicating that the protein was effectively decomposed into peptide segments.

[0153] The enzymatically hydrolyzed solution was centrifuged at a speed of 8500 rpm for 15 minutes. After centrifugation, the supernatant and the precipitate were separated. The supernatant was light yellow in color, highly transparent, and contained a large amount of peptide segments. It will be detected that the α-glucosidase inhibition rate of this supernatant was 55.66%; the α-amylase inhibition rate was 60.46%.

[0154] Inhibitory effects and antioxidant capacities of Xanthoceras sorbifolia Bunge bioactive peptides with different molecular weights on α-glucosidase and α-amylase

[0155] The enzymatic hydrolysate supernatant prepared by the method of Example 2 was separated using ultrafiltration membranes with a molecular weight cut-off of 10 KDa, 3 kDa, and 500 Da to remove macromolecular impurities and small molecules therein. Components with different molecular weight ranges were obtained: <10 KDa (3 kDa - 10 KDa), <3 kDa (500 Da - 3 kDa), and <500 Da. The concentrated solution after ultrafiltration showed a higher peptide concentration and purity.

[0156] The peptide components with different molecular weights after the above ultrafiltration were freeze-dried into dry powders, and then each was prepared into a solution with a concentration of 10 mg / mL. The inhibitory effects of the peptide components with different molecular weights after ultrafiltration on α-glucosidase and α-amylase and their antioxidant capacities were measured, and 10 mg / mL acarbose and 10 mg / mL glutathione (GSH) were used as controls. The results are shown in Table 1.

[0157] Table 1 Inhibitory effects of solutions with different molecular weights after ultrafiltration on α-glucosidase and α-amylase and antioxidant capacities

[0158]

[0159] Note: Different letters in the table indicate significant differences, p < 0.05.

[0160] As can be seen from Table 1, the peptide component with a molecular weight <3 kDa (specific range 500 Da - 3 kDa) showed the best activity performance. Specifically, the inhibition rates of this component on α-glucosidase and α-amylase were as high as 66.12 ± 1.21% and 72.17 ± 0.76% respectively; the antioxidant activities (DPPH and H2O2 scavenging capacities, Fe 3+ reducing capacity) were between 86.67 ± 1.53% - 91.33 ± 1.15%. Given that this component showed excellent inhibitory effects, strong antioxidant activities, and considerable yields. Based on the comprehensive consideration of the above factors, it was decided to select the component with a molecular weight <3 KDa for subsequent experimental studies.

[0161] Hypoglycemic effect of Xanthoceras sorbifolia Bunge active peptides with a molecular weight <3 kDa on diabetic mice

[0162] The diabetes model was established by feeding C57BL / 6J mice with a high-fat diet and injecting streptozotocin. The hypoglycemic activity of the peptide fraction with a molecular weight < 3 kDa (specific range: 500 Da - 3 kDa) obtained by ultrafiltration using the above method was detected in vivo. The experiment was divided into 6 groups in total: CON (control group), MOD (model group), MET (positive drug group, metformin 200 mg / kg), XSBP-H (high-dose group 600 mg / kg), XSBP-M (medium-dose group 400 mg / kg), and XSBP-L (low-dose group 200 mg / kg). Administration was by gavage once a day for 6 weeks of intervention treatment.

[0163] During the administration period, the blood glucose, water intake, body weight, OGTT, and AUC area changes of the mice were detected. The results are as Figures 11 - 15 shown. As Figure 11 can be seen, except for the mice in the control group (CON), the fasting blood glucose values of all experimental group mice increased significantly. As the intervention treatment continued, especially at the sixth week, compared with the model group (MOD), the blood glucose values of the treatment group mice decreased significantly compared with before treatment. Among them, the blood glucose reduction of the mice in the high-dose Xanthoceras sorbifolia peptide (XSBP-H) treatment group was particularly significant, approaching 54%. This obvious blood glucose reduction phenomenon indicates that after a period of continuous treatment, high-dose Xanthoceras sorbifolia peptide can effectively reduce the fasting blood glucose level of type 2 diabetes model mice.

[0164] The results of the water intake of each group of mice are as Figure 12 shown. During the treatment period, the water intake of the mice in the model group (MOD) was significantly higher than that in the control group (CON), which is consistent with the typical symptoms of type 2 diabetes. As the intervention treatment progressed, the water intake of the mice in the very high-dose Xanthoceras sorbifolia peptide group (XSBP-H) and the metformin group (MET) decreased significantly compared with the MOD group, with a decrease of approximately 34% to 40%. This result indicates that high-dose Xanthoceras sorbifolia peptide can significantly relieve the polydipsia symptoms of mice.

[0165] As Figure 13 shown, the body weight change of the mice showed the following trend: compared with the control group (CON), the body weight of the mice in the MOD group gradually decreased; however, after the intervention treatment, the body weight of the mice in the high-dose Xanthoceras sorbifolia peptide (XSBP-H) group gradually increased, showing a significant difference from the mice in the MOD group.

[0166] After 6 weeks of gavage, the oral glucose tolerance test (OGTT) was performed after fasting for 12 hours. By orally administering a glucose solution to the mice and measuring the blood glucose values at 0, 30, 60, 90, and 120 min, the area under the curve AUC was calculated according to the following formula:

[0167]

[0168] In the formula: C0, C1, C2, C3, and C4 are the blood glucose values at 0, 30, 60, 90, and 120 minutes respectively.

[0169] Figure 14 and Figure 15 showed the results of the oral glucose tolerance test (OGTT) in mice and the area under the curve (AUC) of the time-blood glucose curve, which were used to evaluate the ability of pancreatic islet β cells to regulate glucose levels. Compared with the control group (CON), the oral glucose tolerance of the mice in the model group (MOD) was significantly increased, indicating that the diabetic state inhibited the ability of mice to regulate glucose levels. However, in the treatment groups, the AUC of the mice was significantly decreased. Specifically, the AUC of the high-dose Xanthoceras sorbifolia peptide group (XSBP-H) decreased by 25%, and the AUC of the metformin group (MET) decreased by 30%. These results suggest that high-dose Xanthoceras sorbifolia peptide can significantly improve the glucose regulation ability of diseased mice after treatment.

[0170] Mice were anesthetized with isoflurane, and then blood was collected from the orbital cavity, the liver was collected, etc. and stored at -80 °C for subsequent analysis. These samples were centrifuged at 10,000 revolutions per minute for 15 minutes at 4 °C, and then the supernatant was quickly separated. Using an Elisa kit, biochemical indicators such as triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), alanine aminotransferase (ALT), glutathione reductase (GSH), and aspartate aminotransferase (AST) in the supernatant were detected. The judgment basis of the test results was from the operation guide provided by Nanjing Jiancheng Bioengineering Research Institute.

[0171] The results of measuring the blood lipid levels (TG, TC, HDL-C, LDL-C) of mice and the indicators of liver injury (liver glycogen content, GSH, ALT, and AST) are as Figures 16 - 19 shown.

[0172] Due to glucose metabolism disorders, diabetic mice are often accompanied by abnormal lipid metabolism, resulting in increased levels of TG, TC, and LDL-C and decreased levels of HDL-C. The mice in the MOD group showed obvious characteristics ( Figure 16 and Figure 17 ). After intervention treatment, the blood lipid indicators of the mice were improved. Compared with the mice in the MOD group, the levels of TG, TC, and LDL-C in the XSBP-H group and the MET group were significantly decreased, and the reduction ranges of each index were 20%-35% and 19%-45% respectively. Moreover, the levels of HDL-C were also significantly increased by about 39% and 48%, further indicating that high-dose Xanthoceras sorbifolia peptide can relieve the lipid metabolism disorder in the liver.

[0173] Figure 18 and Figure 19 The effects on liver injury indicators. Compared with the control group (CON), the livers of mice in the model group (MOD) showed significant biochemical changes. Specifically, the glycogen content and glutathione (GSH) content in the livers of MOD group mice decreased, indicating that the energy storage capacity and antioxidant defense mechanism of their livers might be damaged; at the same time, the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents in the sera of MOD group mice increased significantly, which are usually regarded as signs of damaged liver cells. After intervention treatment, the glycogen content (Hepatic glycogen content) and glutathione (GSH) content in the livers of XSBP-H group mice increased significantly; and the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents decreased significantly, further indicating that the high-dose Xanthoceras sorbifolia peptide of the present invention has a good effect on protecting the liver.

[0174] Mouse liver tissues were taken for HE staining and liver oil red staining observations, and the results are as Figure 20 and Figure 21 shown.

[0175] Under HE staining observation ( Figure 20 ), compared with the control group (CON), the hepatocytes of mice in the model group (MOD) showed obvious swelling, the liver tissue structure became disordered, with obvious inflammatory infiltration, and a large number of vacuoles of different sizes could be seen around the cell nuclei. These changes all indicated that the liver cells of MOD group mice were damaged. However, after intervention treatment with high-dose Xanthoceras sorbifolia peptide (XSBP-H) and metformin (MET), the hepatocytes of mice showed an improved trend. Especially in the XSBP-H group mice, the number of vacuoles around the liver cell nuclei decreased significantly, and the structure of hepatocytes gradually tended to be normal, indicating that Xanthoceras sorbifolia peptide has an obvious protective effect on the liver cells of diabetic mice.

[0176] As Figure 21 shown, it is the oil red staining result of the mouse liver, and the corresponding fat area statistics are presented in Figure 22 In Figure 21Among them, the red area represents the fat accumulated in the tissue. It is worth noting that almost no red fat was shown in the liver sections of the mice in the CON group, while the red area in the sections of the mice in the MOD group increased significantly. This change intuitively reflects that diabetes can cause metabolic disorders in the livers of mice, which in turn leads to a large accumulation of fat. After the intervention treatment, the fat accumulation in the livers of the mice was significantly improved. Specifically, the red area shown by Oil Red staining was significantly reduced. Among them, in the mice in the XSBP-H group, the positive area of Oil Red staining decreased by 40%. This data further verifies that high-dose Xanthoceras sorbifolia Bunge peptide has a significant improvement effect on the fat accumulation in the livers of diabetic mice.

[0177] Effect of Xanthoceras sorbifolia Bunge active peptide with molecular weight <3 kDa on the intestinal flora of diabetic mice

[0178] Total DNA of the mouse fecal cecal contents of each group (CON, MOD, and XSBP-H) was extracted according to the instructions of the Omega Bio-Tek kit PF Mag-Bind Stool DNA Kit. The concentration and purity of the extracted DNA were detected by 1% agarose gel electrophoresis. The V3-V4 region of bacteria was amplified by PCR using primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’), and the PCR products were detected and quantified using a Quantus TM Fluorometer. The library was constructed using the NEXTFLEX Rapid DNA-Seq Kit library construction kit, and sequencing was performed through the Illumina PE300 / PE250 platform.

[0179] Sequencing data evaluation: Rank-abundance curves and rarefaction curves were selected to evaluate the species abundance and evenness of the measured samples. The rank-abundance curve reflects the richness and evenness of the species contained in each sample; the richer the species, the wider the curve in the horizontal coordinate direction; the more even the species, the flatter the curve. As Figure 23 can be seen, the horizontal zigzag lines of the samples in each group are relatively wide and finally tend to be flat, indicating that the sample composition is uniform and rich. The rarefaction curve is obtained by statistically analyzing the microbial diversity index of the flora samples at different sequencing depths. As Figure 24 can be seen, as the sequencing depth increases, the number of OTUs also gradually increases and finally tends to be flat, indicating that the data volume is relatively reasonable.

[0180] Overall sample microbial diversity OTUs analysis: To study the species composition of each sample, all samples were clustered by OTUs (Operational Taxonomic Units) and species annotation was performed. A Venn graph (VennGraph) was drawn. The results are as Figure 25 shown: There are 52 OTUs shared by the CON group, MOD group, and XSBP-H group, 274 unique OTUs in the MOD group, and 1312 unique OTUs in the XSBP-H group. This suggests that type 2 diabetes can cause changes in the number of OTUs compared to normal group mice.

[0181] α-diversity analysis: Ace, Chao1, and Shannon are important components of the α-diversity index. As Figure 26 shown, compared with the CON group, the Ace, Simpson, and Chao1 indices in the MOD group all decreased, and there were significant differences (p<0.01), indicating that diabetes can reduce the intestinal microbial diversity of mice and cause dysregulation of the flora; compared with the MOD group, the Shannon, Chao1, and Ace indices in the XSBP-H group were significantly increased, indicating that high-dose Xanthoceras sorbifolia Bunge peptide can effectively counteract the reduction of intestinal flora diversity caused by diabetes and help maintain the homeostasis of the intestinal flora.

[0182] β-diversity analysis: β-diversity is an index that measures the species diversity differences between different microbial communities. It reveals the differences in microbial community structure between samples by comparing the species composition between communities. To visually display these differences, principal coordinate analysis (PCoA) and non-metric multidimensional scaling analysis (NMDS) can be used to reflect the differences between samples. In the PCoA and NMDS graphs, the distance between samples reflects their similarity in composition and abundance. Specifically, the smaller the distance between samples, the more similar their microbial community composition and species abundance are. As Figure 27 、 Figure 28 shown, good discrimination was shown among the CON group, MOD group, and XSBP-H group, indicating that diabetes can affect the composition of the intestinal flora of mice. At the same time, high-dose Xanthoceras sorbifolia Bunge peptide has the ability to regulate the structural composition and diversity of the intestinal flora in diabetic mice, thus playing a preventive and protective role against the intestinal flora imbalance caused by diabetes in mice.

[0183] Community composition analysis and species difference analysis: The intestinal flora abundances of mice in each group were statistically analyzed at the phylum and genus levels to analyze their flora compositions. As Figure 30As shown, the dominant phyla in the mouse intestine are Firmicutes, Bacteroidota, and Proteobacteria, etc. Compared with the CON group, the relative abundances of Firmicutes and Proteobacteria in the MOD group increased, from 24.93% to 40.43% and from 1.41% to 20.27% respectively; the relative abundance of Bacteroidota decreased, from 67.34% to 27.31%. After intervention, the proportion of Bacteroidota increased, and it had an inhibitory effect on the growth of Firmicutes. Compared with the MOD group, the proportion of Firmicutes in the XSBP-H group decreased to 32.09%, and the proportion of Bacteroidota increased to 54.78%.

[0184] Human and animal studies have shown that the ratio of Firmicutes to Bacteroidetes (F / B ratio) or the ratio of Bacteroidetes to Firmicutes (B / F ratio) is an indicator of intestinal microbiota health. As Figure 31 shown, the F / B value of the intestinal microbiota of mice in the MOD group decreased significantly. However, after intervention treatment, the F / B values of mice in the XSBP-H group and the MET group both increased.

[0185] In summary, high-dose XSBP-H of Xanthoceras sorbifolia Bunge peptide has a regulatory effect on the intestinal microbiota disorder caused by diabetes in mice, and indirectly affects the regulation of blood glucose by intestinal microorganisms through the regulation of the intestinal microbiota.

[0186] The above description is only a preferred embodiment of the present invention, and it is not a limitation of the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal, characterized in that, The steps are as follows: (1) Dissolve the defatted Xanthoceras sorbifolia Bunge meal powder in deionized water according to a mass ratio of 1:10 to 1:30, and stir evenly to obtain a defatted Xanthoceras sorbifolia Bunge powder solution; (2) Pretreatment: Place the defatted Xanthoceras sorbifolia Bunge powder solution in a water bath, perform high-temperature heat treatment at 50 - 100 °C for 10 - 50 minutes, and then perform ultra-high pressure pretreatment at 150 - 350 MPa for 150 - 350 s; (3) Enzymolysis: Add protease to the pretreated solution, mix evenly, perform enzymolysis at 40 - 60 °C for 1 - 5 hours, with a pH of 6 - 10; Centrifuge the enzymolyzed solution and take the supernatant to obtain the product.

2. The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal according to claim 1, characterized in that, The protease is at least one of alkaline protease, compound protease, and flavor protease; The addition amount of the protease is 3000 - 5000 U / g.

3. The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia meal according to claim 2, characterized in that, The protease is alkaline protease, compound protease, and flavor protease; The addition amount of each protease is 4500 U / g.

4. The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal according to claim 1, characterized in that, In the step (1), the mass ratio of the defatted Xanthoceras sorbifolia Bunge meal powder to deionized water is 1:

10.

5. The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal according to claim 1, characterized in that, In the step (2), the high-temperature heat treatment temperature is 80 °C, and the treatment time is 30 min.

6. The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia meal according to claim 1, characterized in that, In the step (2), the ultra-high pressure pretreatment pressure is 200 MPa, and the pretreatment time is 350 s.

7. The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolia Bunge meal according to claim 1, characterized in that, In the step (3), the enzymolysis time is 4 hours, and the temperature is 50 °C.

8. The method for extracting bioactive peptides with antioxidant and hypoglycemic effects from Xanthoceras sorbifolium Bunge meal according to any one of claims 1 to 7, characterized in that, It further includes the following steps: Perform molecular weight fractionation on the supernatant obtained in step (3) through an ultrafiltration centrifuge tube, and collect the fraction with a molecular weight of 500 Da - 3 kDa.

9. Use of the bioactive peptide prepared by the method according to claim 8 and having antioxidant and hypoglycemic effects in the preparation of a product having antioxidant and hypoglycemic effects, characterized in that, The product is a food, drug, or health product.

10. The application according to claim 9, wherein The antioxidant is DPPH scavenging activity, hydroxyl ion radical scavenging activity, and ferric ion reducing ability; The blood sugar lowering effect is to have the activity of inhibiting α-glucosidase and inhibiting α-amylase.

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