Preparation method of mung bean sprout dietary fiber capable of improving insulin resistance activity

Abstract

The invention belongs to the technical field of polysaccharides, and particularly relates to a preparation method and application of mung bean sprout dietary fibers. The monosaccharide of the dietary fiber mainly comprises fucose, arabinose, galactose, glucose and mannose, and the molar ratio of the fucose to the arabinose to the galactose to the glucose to the mannose is 12.28: 2.60: 27.59: 7.06: 50.47. The structural unit is formed by mutually connecting-> 6)-alpha-D-Glcp-(1->,-> 5)-alpha-L-Araf-(1->,-> 3, 6)-alpha-D-Glcp-(1-> and the like to form a main chain, and a branch chain is mainly formed by connecting beta-D-Manp-(1-> to an O-3 position of a sugar residue-> 3, 6)-alpha-D-Glcp-(1-> and the like. In-vitro cell experiments prove that the dietary fiber can improve the insulin resistance state of HepG2 cells, the glucose consumption and the cell glycogen synthesis effect are improved, the activity of hexokinase and pyruvate kinase is improved, and the dietary fiber has no toxic effect influence on the cell growth activity in vitro. Therefore, the dietary fiber has a potential effect of improving insulin resistance, and is expected to become a functional food raw material for treating diabetes.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide research and relates to a method for preparing mung bean sprout dietary fiber with activity of improving insulin resistance. Background Art

[0002] Insulin is produced and secreted by the beta cells of the pancreas and is a key hormone that regulates glucose homeostasis, protein synthesis, lipid metabolism, and cell growth. Insulin resistance is the main factor causing metabolic syndromes such as type II diabetes, atherosclerosis, or non-alcoholic fatty liver disease, which is manifested as a decrease in cell sensitivity to insulin and the inability of glucose in the blood to be taken up and consumed in a timely manner. The body is in a state of hyperglycemia for a long time, which stimulates the pancreas to continuously secrete insulin, causing islet dysfunction. Therefore, improving insulin sensitivity is the key to controlling insulin resistance and preventing metabolic diseases. Excessive levels of glucose and insulin in the blood, increased free fatty acids, and oxidative stress can all cause insulin resistance in liver cells. Long-term use of drugs to lower blood sugar will produce drug resistance and have certain side effects on the human body. Therefore, it is of great significance to find a safe hypoglycemic substance.

[0003] Mung bean is an annual herbaceous food crop of the genus Vigna in the Leguminosae family. Its planting area and yield are among the highest in the world, and it has high nutritional and medicinal value. Mung bean contains rich nutrients and bioactive substances, such as polyphenols, peptides, dietary fiber, unsaturated fatty acids, etc., and has the effects of reducing swelling and diuresis, lowering blood pressure and improving eyesight, clearing away heat and detoxifying. Dietary fiber is an important active substance in mung bean, which has antioxidant, immunomodulatory, and lipid metabolism-improving effects. Studies have shown that salt stress germination can change the content and biological activity of nutritional components in grains, and is a potential processing method to improve the biological activity of functional components. However, the effect of salt stress germination treatment on the structure and biological activity of mung bean dietary fiber is still unclear. Summary of the invention

[0004] The present invention adopts a green and efficient dietary fiber extraction process and method, using mung beans as raw materials, and obtains a dietary fiber (named SMDF-1 in the text) through salt stress germination, enzyme-assisted extraction, and step-by-step purification. In vitro experiments show that SMDF-1 has the ability to inhibit α-glucosidase activity and improve the insulin resistance activity of HepG2 cells, and is expected to be developed into a functional food raw material for lowering blood sugar.

[0005] One of the purposes of the present invention is to provide a mung bean sprout dietary fiber.

[0006] A second object of the present invention is to provide a method for preparing mung bean sprout dietary fiber.

[0007] The third object of the present invention is to provide a use of the mung bean sprout dietary fiber in a functional food for improving insulin resistance.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following experimental technical scheme is adopted:

[0009] The dietary fiber of the invention mainly consists of fucose, arabinose, galactose, glucose and mannose.

[0010] The unit structure of the dietary fiber is composed of →6)-α-D-Glcp-(1→, →5)-α-L-Araf-(1→ and →3,6)-α-D-Glcp-(1→, etc. connected to each other to form a main chain, and the branch chain is mainly composed of β-D-Manp-(1→ connected to the O-3 position of the sugar residue →3,6)-α-D-Glcp-(1→, etc.

[0011] The weight average molecular weight of the salt-stressed mung bean sprout dietary fiber is in the range of 100-200 kDa.

[0012] Preferably, the salt stress mung bean sprout dietary fiber has Figure 1 High performance gel permeation chromatography (HPGPC) diagram shown.

[0013] Preferably, the salt stress mung bean sprout dietary fiber has Figure 2 The infrared spectrum is consistent with the absorption peak of the main functional groups in 3236cm -1 OH stretching vibration, 2900 cm -1 The CH stretching vibration is at 1409 cm -1 The methylene CH shear vibration is at 2100 cm -1 is CH stretching vibration, 1634cm -1 The stretching vibration of C=C is 1144cm -1 、1073cm -1 、1019cm -1 is CO stretching vibration, 840cm -1 The peak indicates the presence of α-glycosidic bond.

[0014] Preferably, the salt stress mung bean sprout dietary fiber has Figure 3 The monosaccharide composition chromatogram of FIG. The molar ratio of fucose, arabinose, galactose, glucose and mannose is 10-15: 2-5: 25-30: 5-10: 45-55, preferably 12.28: 2.60: 27.59: 7.06: 50.47.

[0015] Preferably, the salt stress mung bean sprout dietary fiber has Figure 4 The scanning electron microscope image shows a smooth surface and blocky structure.

[0016] Preferably, the salt stress mung bean sprout dietary fiber has Figure 5 The total ion current of methylation analysis in is composed of t-Ara(f), t-Xyl(p), t-Man(p), 5-Ara(f), 2-Ara(p), 6-Glc(p), 4-Gal(p), 2,5-Ara(f), 3,6-Glc(p), 4,6-Gal(p).

[0017] Preferably, the salt stress mung bean sprout dietary fiber has Figure 6 The main signal values ​​in the one-dimensional NMR spectrum are consistent. The δ5.33 / 99.29 signal belongs to →6)-α-D-glucose-(1→; δ4.91 / 98.22 signal belongs to β-D-mannose-(1→; δ5.02 / 107.25 signal belongs to →5)-α-L-arabinose-(1→; δ5.29 / 99.71 signal belongs to →3,6)-α-D-Glcp-(1→.

[0018] In another aspect, the present invention provides a method for preparing the salt-stressed mung bean sprout dietary fiber (SMDF-1), comprising the following steps:

[0019] a. Salt stress treatment of germinating mung beans:

[0020] Take fresh mung beans with full grains, sterilize, stimulate, soak the seeds, and place them in a constant temperature and humidity incubator in the dark.

[0021] Preferably, step a comprises: taking fresh mung beans with full seeds, disinfecting: soaking in 3% to 5% 84 disinfectant for half an hour, and rinsing with distilled water until there is no residue. Stimulating seeds: soaking mung beans in water at 40°C to 60°C for 2 minutes. Soaking seeds: soaking mung beans in water at 25°C to 30°C for 6 to 8 hours. Spread the treated mung beans evenly on a tray, pour in a NaCl solution with a concentration range of 0 to 0.15M, replace the culture solution every 12 hours, and place in a constant temperature and humidity incubator at 25°C and 70% humidity to avoid light for culture.

[0022] Further preferably, step a comprises: taking fresh mung beans with full seeds, disinfecting: soaking in 5% 84 disinfectant for half an hour, and rinsing with distilled water until no residue is left. Stimulating seeds: soaking mung beans in 50°C water for 2 minutes. Soaking seeds: soaking mung beans in 27°C water for 8 hours. The treated mung beans are evenly spread on a tray, poured with a 0.05M NaCl solution, the culture solution is replaced every 12 hours, and placed in a constant temperature and humidity incubator at 25°C and 70% humidity to avoid light for culture.

[0023] b.Dietary fiber extraction:

[0024] The dried mung bean sprouts are crushed, and then the fat is removed and then enzymatic extraction is performed. The obtained extract is concentrated, dialyzed, and centrifuged to remove the precipitate. The obtained supernatant is precipitated with alcohol and centrifuged, and the obtained precipitate is washed and dried to obtain enzyme-extracted salt-stressed mung bean sprout dietary fiber;

[0025] Preferably, the step b comprises: taking dried mung bean sprouts, crushing them, adding 5 to 8 times the weight of anhydrous ethanol to remove small molecules such as lipids, adding distilled water to the defatted sample powder at a solid-liquid ratio of 1:20 to 1:30, adjusting the pH value to 6 to 7, adding 1% to 2% α-amylase and saccharifying enzyme, extracting at 60° C. for 3 to 5 hours, combining and concentrating the extracts, dialyzing, concentrating again, centrifuging to obtain a supernatant, adding anhydrous ethanol about 4 to 6 times the volume of the supernatant, centrifuging to obtain a precipitate, and vacuum freeze-drying to obtain enzyme-extracted salt-stressed mung bean sprouts crude dietary fiber.

[0026] Further preferably, the step b comprises: taking dried mung bean sprouts, crushing them, adding 6 times the weight of anhydrous ethanol to remove small molecules such as lipids, adding distilled water to the defatted sample powder at a solid-liquid ratio of 1:25, adjusting the pH to 6.5, adding 1% α-amylase and saccharifying enzyme, extracting at 60°C for 4 hours, combining and concentrating the extracts, dialyzing, concentrating again, centrifuging to obtain a supernatant, adding anhydrous ethanol 4 times the volume of the supernatant, centrifuging to obtain a precipitate, and vacuum freeze-drying to obtain enzyme-extracted salt-stressed mung bean sprouts crude dietary fiber.

[0027] c. Dietary fiber purification:

[0028] c1. The enzyme extract prepared in step b was dissolved in water for crude dietary fiber from salt-stressed mung bean sprouts, and then purified by an anion exchange column, eluted with water and 0.2 to 0.6 M NaCl solution, and the eluted fractions were collected to obtain salt-stressed mung bean sprouts dietary fiber MDF-0;

[0029] Preferably, the step c1 comprises: taking the enzyme-extracted salt-stressed mung bean sprouts crude dietary fiber prepared in step b, adding about 10 to 15 times the weight of water to dissolve it, separating it through an anion exchange column, eluting it with deionized water, 0.2M, 0.4M and 0.6M NaCl solutions in sequence, detecting it with sulfuric acid-phenol, collecting and merging the eluate of deionized water according to the elution curve, concentrating, centrifuging, taking the supernatant for dialysis, and freeze-drying it to obtain the preliminarily purified dietary fiber MDF-0;

[0030] Further preferably, the step c1 comprises: taking the enzyme-extracted salt-stressed mung bean sprouts crude dietary fiber prepared in step b, adding about 10 times the weight of water to dissolve it, separating it through a DEAE cellulose DE-52 anion exchange column, eluting it with deionized water, 0.2M, 0.4M and 0.6M NaCl solution in sequence, detecting it with sulfuric acid-phenol, collecting and merging the eluate of deionized water according to the elution curve, concentrating, centrifuging, taking the supernatant for dialysis, and freeze-drying it to obtain the preliminarily purified dietary fiber MDF-0;

[0031] c2. The dietary fiber MDF-0 prepared in step c1 was dissolved in about 80 to 100 times the weight of deionized water, purified by gel chromatography, eluted with deionized water, and the sulfated polysaccharide SMDF-1 component was collected and combined according to the elution curve to obtain the salt-stressed mung bean sprout dietary fiber SMDF-1;

[0032] Preferably, the step c2 comprises: dissolving the dietary fiber MDF-0 prepared in step c1 in about 100 times the weight of deionized water, purifying through a Sephadex G-100 polysaccharide gel column, eluting with deionized water, detecting by a sulfuric acid-phenol method, collecting and merging the main component of dietary fiber SMDF-1 according to the elution curve, concentrating, dialyzing, and freeze-drying to obtain the salt-stressed mung bean sprout dietary fiber SMDF-1.

[0033] Structural identification of dietary fiber: The structure of the salt-stressed mung bean sprout dietary fiber SMDF-1 was determined by comprehensive analysis of its monosaccharide composition, molecular weight, scanning electron microscopy, methylation, infrared and nuclear magnetic resonance.

[0034] Another aspect of the present invention provides use of the dietary fiber or the pharmaceutical composition in preparing a medicament for treating and / or preventing insulin resistance.

[0035] The insulin resistance is preferably insulin-induced HepG2 cell insulin resistance.

[0036] The technical solution of the present invention has at least the following technical effects:

[0037] The present invention prepares salt-stressed mung bean sprout dietary fiber containing specific monosaccharide composition, molecular weight and bonding structure through specific processing, extraction and separation methods. In vitro experiments have shown that the dietary fiber SMDF-1 can increase the glucose consumption and glycogen synthesis of insulin-resistant HepG2 cells, activate hexokinase and pyruvate kinase, and has no effect on cell growth vitality in vitro, and has great application prospects in candidate drugs for preventing and / or treating insulin resistance.

[0038] The present invention has been described in detail above, but the above embodiments are only illustrative in nature and do not limit the present invention. In addition, this article is not limited by any theory described in the above prior art or invention content or the following examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 High performance gel permeation chromatography (HPGPC) of dietary fiber SMDF-1 of mung bean sprouts under salt stress

[0040] Figure 2 Characteristic infrared spectrum of SMDF-1 dietary fiber of mung bean sprouts under salt stress

[0041] Figure 3 The monosaccharide composition of SMDF-1 dietary fiber from mung bean sprouts under salt stress

[0042] Figure 4 Scanning electron micrographs of dietary fiber SMDF-1 from mung bean sprouts under salt stress (A) ×1000, (B) ×2000

[0043] Figure 5 Total ion current for methylation analysis of SMDF-1 in mung bean sprouts under salt stress

[0044] Figure 6 One-dimensional NMR spectrum of dietary fiber SMDF-1 from mung bean sprouts under salt stress (A) 1 H NMR, (B) 13 C NMR spectrum

[0045] Figure 7 The hydrogen-hydrogen correlation 2D NMR spectrum of SMDF-1, a dietary fiber of mung bean sprouts subjected to salt stress, (A) COSY, (B) NOESY

[0046] Figure 8 The carbon-hydrogen correlation 2D NMR spectrum of SMDF-1, a dietary fiber from mung bean sprouts subjected to salt stress, (A) HSQC, (B) HMBC, (C) chemical structure

[0047] Fig. 9 Effect of salt-stressed mung bean sprout dietary fiber SMDF-1 prepared in Example 1 on the survival rate of HepG2 cells

[0048] Fig.10 Effect of salt-stressed mung bean sprout dietary fiber SMDF-1 prepared in Example 1 on glucose consumption of IR-HepG2 cells

[0049] Fig.11 Effect of salt-stressed mung bean sprout dietary fiber SMDF-1 prepared in Example 1 on glycogen synthesis in IR-HepG2 cells

[0050] Fig.12 Effects of salt-stressed mung bean sprout dietary fiber SMDF-1 prepared in Example 1 on HK (A) and PK (B) activities in IR-HepG2 cells DETAILED DESCRIPTION

[0051] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only for illustrative purposes and do not constitute a limitation on the scope of protection claimed for the present invention.

[0052] Unless otherwise specified, the experimental methods used in the examples are all conventional methods.

[0053] Material:

[0054] Mung beans come from the Institute of Economic Crops of Jiangsu Academy of Agricultural Sciences and are produced in Jiangsu, China.

[0055] equipment:

[0056] High performance gel permeation chromatography was performed using a tsl-gelG3000SWXL column (TSK, Japan) and a 1200 high performance liquid chromatograph with a differential detector (Agilent, USA).

[0057] The monosaccharide composition was determined using ICS 5000+ ion chromatography (Thermo Fisher Scientific, USA).

[0058] The surface structure was determined using an EVO-LS10 scanning electron microscope (Carl Zeiss AG, Germany).

[0059] Infrared analysis was performed using a Nicolet iS50 Fourier transform infrared spectrometer (Thermo Fisher Scientific (China) Co., Ltd.).

[0060] The nuclear magnetic resonance analysis was performed using a Brucker AM-500 nuclear magnetic resonance spectrometer (Brucker, Germany).

[0061] Preparation Example 1: Preparation of Salt-stressed Mung Bean Sprout Dietary Fiber SMDF-1

[0062] a. Mung bean germination under salt stress and dietary fiber extraction:

[0063] Take fresh mung beans with full seeds, soak them in 5% 84 disinfectant for half an hour for disinfection, rinse with distilled water until there is no residue, soak them in 50℃ water for 2 minutes, soak them in 27℃ water for 8 hours, spread them evenly on a tray, pour in 0.05M NaCl solution, replace the culture medium every 12 hours, and place them in a constant temperature and humidity incubator at 25℃ and 70% humidity in the dark for culture.

[0064] Take dried mung bean sprouts, crush them, add 6 times the weight of anhydrous ethanol to remove small molecules such as lipids, add distilled water to the defatted sample powder at a solid-liquid ratio of 1:25, adjust the pH to 6.5, add 1% α-amylase and saccharifying enzyme, extract at 60°C for 4 hours, combine and concentrate the extracts, dialyze, concentrate again, centrifuge to obtain the supernatant, add anhydrous ethanol 4 times the volume of the supernatant, centrifuge to obtain the precipitate, and vacuum freeze-dry to obtain the enzyme-extracted salt-stressed mung bean sprouts crude dietary fiber.

[0065] b. Purification of dietary fiber SMDF-1:

[0066] Take the above-prepared enzyme-extracted salt-stressed mung bean sprouts crude dietary fiber, add about 10 times the weight of water to the enzyme-extracted salt-stressed mung bean sprouts crude dietary fiber to dissolve, separate through DEAE cellulose DE-52 anion exchange column, sequentially elute with deionized water, 0.2M, 0.4M and 0.6M NaCl solution, detect with sulfuric acid-phenol, collect and merge the eluate of deionized water according to the elution curve, concentrate, centrifuge, dialyze the supernatant, freeze-dry, and obtain the preliminary purified dietary fiber MDF-0. MDF-0 is dissolved in about 100 times the weight of deionized water, purified through Sephadex G-100 dextran gel column, eluted with deionized water, detect with sulfuric acid-phenol method, collect and merge the main component of dietary fiber SMDF-1 according to the elution curve, concentrate, dialyze, freeze-dry, and obtain the salt-stressed mung bean sprouts dietary fiber SMDF-1.

[0067] c. Structural identification and analysis of dietary fiber SMDF-1:

[0068] (1) The relative molecular weight of dietary fiber SMDF-1 was determined by high performance gel permeation chromatography (HPGPC) to be 165 kDa, and its purity was 97% (e.g. Figure 1 ).

[0069] (2) The monosaccharide composition of the hydrolyzed sample of dietary fiber SMDF-1 was measured by ion chromatography, and it was determined that it mainly contained fucose, arabinose, galactose, glucose and mannose, with a molar ratio of 0.48:3.95:3.86:82.69:9.02.

[0070] (3) Glucose in dietary fiber SMDF-1 exists in two bond types: 6-Glc(p) and 3,6-Glc(p), with a ratio of about 18:1, accounting for 62.81% of the total, indicating that the Glc(p) residue is the main structural unit. Arabinose exists in three bond types, namely t-Ara(f), 5-Ara(f) and 2-Ara(p), accounting for 30.39%. Galactose exists in two bond types: 4-Gal(p) and 4,6-Gal(p), accounting for about 3.69%. Mannose exists in the t-Man(p) bond type, accounting for about 3.10%.

[0071] (4) Infrared spectrum (such as Figure 3 ), 3236cm -1 OH stretching vibration, 2900 cm -1 The CH stretching vibration is at 1409 cm -1 The CH shear vibration is at 2100cm -1 The stretching vibration of CH is at 1634 cm -1 The stretching vibration of C=C is 1144cm -1 、1073cm -1 、1019cm -1 It is CO stretching vibration. 840cm -1 The α-type glycosidic bond was located in the residue, indicating that SMDF-1 was mainly composed of pyranose rings.

[0072] (5) NMR analysis: A dietary fiber SMDF-1 solution with a concentration of 40 mg / mL was prepared using D2O as solvent, and the one-dimensional and two-dimensional NMR spectra were measured at 25°C using a 500 MHz NMR spectrometer, and the structure was characterized.

[0073] The SMDF-1 hydrogen spectrum signal is mainly concentrated between δ3.0 and 5.5 ppm, and the chemical shifts of the anomeric hydrogen are δ4.91, 5.02, 5.29, and 5.33 ppm, respectively, indicating that it contains a variety of sugar residues. SMDF-1 recognizes multiple signal peaks in the anomeric carbon region, combined with 13 The cross peaks in the anomeric region of the C NMR spectrum and the HSQC spectrum confirmed the existence of anomeric signals as follows: δ 5.33 / 99.29, 4.91 / 98.22, 5.02 / 107.25, 5.29 / 99.71 ppm, which were recorded as sugar residues A, B, C, and D, respectively.

[0074] Table 1 represents the signal assignment of hydrocarbons

[0075]

[0076] The anomeric signal 85.33 / 99.29ppm (H1 / C1) indicates that residue A may be an α-configuration glucose residue. Figure 7 A), based on the cross peaks, H2, H3, H4, H5, and H6 of residue A were determined to be 3.53 ppm, 3.2 ppm, 3.69 ppm, 3.37 ppm, and 3.80 / 3.68 ppm, respectively. By HSQC signal attribution ( Figure 8 A) The chemical shifts of C on the sugar ring: C1, C2, C3, C4, C5, and C6 are 99.29ppm, 74.21ppm, 74.08ppm, 72.07ppm, 71.06ppm, and 68.87ppm, respectively. The chemical shifts of C1 and C6 shift to the low field, indicating that the residue is substituted at the O-1 and O-6 positions of the sugar ring. It is inferred that the sugar residue A may be →6)-α-D-glucose-(1→

[0077] The anomeric signal δ4.91 / 98.22ppm (H1 / C1) indicates that residue B may be a β-configuration mannose residue. Figure 7 A), based on the cross peaks, H2, H3, H4, H5, and H6 of residue B were determined to be 3.75 ppm, 3.48 ppm, 3.67 ppm, 3.89 ppm, and 3.77 / 4.02 ppm, respectively. By HSQC signal assignment ( Figure 8 A) Chemical shifts of C on the sugar ring: C1, C2, C3, C4, C5, and C6 are 98.22ppm, 74.18ppm, 72.68ppm, 72.23ppm, 73.27ppm, and 60.24ppm, respectively. The chemical shift of C1 shifts to the low field, indicating that the residue is substituted at the O-1 position of the sugar ring. It is inferred that the sugar residue B may be β-D-mannose-(1→

[0078] The anomeric signal δ5.02 / 107.25ppm (H1 / C1) indicates that residue C may be an α-arabinose residue. Figure 7 A), based on the cross peaks, H2, H3, H4, and H5 of residue C were determined to be 4.06 ppm, 3.61 ppm, 3.81 ppm, and 3.70 / 3.94 ppm, respectively. Then, the HSQC signal was assigned ( Figure 8 A) Chemical shifts of C on the sugar ring: C1, C2, C3, C4, and C5 are 107.25ppm, 80.72ppm, 78.57ppm, 80.71ppm, and 69.21ppm, respectively. The chemical shifts of C1 and C5 shift to the low field, indicating that the residue is substituted at the O-1 and O-5 positions of the sugar ring. It is inferred that the sugar residue C may be →5)-α-L-arabinose-(1→

[0079] The anomeric signal δ5.29 / 99.71ppm (H1 / C1) indicates that residue D may be an α-configuration glucose residue. Figure 7 A), based on the cross peaks, H2, H3, H4, H5, and H6 of residue D were determined to be 3.49 ppm, 3.9 ppm, 3.56 ppm, 3.76 ppm, and 3.35 / 3.94 ppm, respectively. Then, the HSQC signal was assigned ( Figure 8 A) Chemical shifts of C on the sugar ring: C1, C2, C3, C4, C5, and C6 are 99.71ppm, 70.77ppm, 77.56ppm, 72.72ppm, 70.08ppm, and 69.63ppm, respectively. The chemical shifts of C1, C3, and C6 shift to the low field, indicating that the residue is substituted at the O-1, O-3, and O-6 positions of the sugar ring. It is inferred that the sugar residue D may be (1→3,6)-α-D-glucose-(1→

[0080] According to the sugar residues 13 C and 1 The chemical shift of H, combined with HMBC ( Figure 8 B) and NOESY ( Figure 7 B) Spectral analysis shows the structures and connection modes in SMDF-1. According to the HMBC spectrum, H1 of sugar residue A and C6 of sugar residue A have a cross peak of δ5.33 / 68.87ppm, and H1 of sugar residue A and C6 of sugar residue D have a cross peak of δ5.33 / 69.63ppm. According to the NOESY spectrum, H1 of sugar residue A and H6 of sugar residue A have a cross peak of δ5.33 / 3.8ppm, H1 of sugar residue A and H5 of sugar residue C have a cross peak of δ5.33 / 3.94ppm, H1 of sugar residue B and H3 of sugar residue D have a cross peak of δ4.91 / 3.9ppm, H1 of sugar residue C and H6 of sugar residue A have a cross peak of δ5.02 / 3.8ppm, and H1 of sugar residue D and H6 of sugar residue A have a cross peak of δ5.29 / 3.8ppm.

[0081] Based on the analysis of one-dimensional and two-dimensional NMR information and methylation results, it was inferred that SMDF-1 is mainly composed of →6)-α-D-Glcp-(1→, →5)-α-L-Araf-(1→ and →3,6)-α-D-Glcp-(1→, etc., which are connected to each other to form the main chain, and the side chain is mainly composed of β-D-Manp-(1→ connected to the O-3 position of the sugar residue →3,6)-α-D-Glcp-(1→, etc. Figure 8 C).

[0082] Test Example 2: Activity of mung bean sprout dietary fiber SMDF-1 in improving insulin resistance

[0083] (1) CCK-8 assay to detect the toxic effect of SMDF-1 on HepG2 cells

[0084] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum (purchased from Gibco, USA), 100 U / mL penicillin and 100 μg / mL streptomycin in a volume ratio of 1:1. HepG2 cells (1×10 5 / well) in a 96-well plate, set up three groups of parallel experiments, and cultured in an incubator at 37°C and 5% CO2 for 24 hours; removed the cell supernatant, added 100μL of mung bean sprout dietary fiber SMDF-1 with a concentration of 0.0625, 0.125, 0.25, 0.5, and 1mg / mL, and cultured for 24 hours, then added 10μL of CCK8 solution for 30 minutes, and the absorbance was detected at 450nm with an enzyme marker. Cell growth activity was calculated according to the following formula: Cell activity = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%. The results are as follows Fig. 9 As shown, it indicates that SMDF-1 has no toxic effect on cells.

[0085] (2) Effect of SMDF-1 on glucose consumption in insulin-resistant HepG2 cells

[0086] HepG2 cells in the logarithmic growth phase were taken and the cell suspension obtained after trypsin digestion was diluted with complete medium to adjust the cell density to 1×10 5 / mL, inoculated in 96-well plates, 100μL per well, the cells were divided into normal group, insulin resistance group, positive control group, and drug group. Except for the normal group, 10 -8 mol / L insulin, acting for 48 hours to induce insulin resistance in HepG2 cells. After modeling, complete culture medium was added to the normal group and insulin resistance group, 35μg / mL metformin hydrochloride solution was added to the positive control group, and 30, 62.5, and 125μg / mL SMDF-1 solutions were added to the drug group. After 24 hours of culture, the glucose consumption of cells in each group was detected. The results are shown in Fig.10 As shown, the glucose consumption of insulin-resistant cells after SMDF-1 intervention was significantly increased.

[0087] (3) Effect of SMDF-1 on glycogen synthesis in insulin-resistant HepG2 cells

[0088] HepG2 cells in the logarithmic growth phase were taken and the cell suspension obtained after trypsin digestion was diluted with complete medium to adjust the cell density to 1×10 5 / mL, inoculated in 96-well plates, 100μL per well, the cells were divided into normal group, insulin resistance group, positive control group, and drug group. Except for the normal group, 10 -8 mol / L insulin, acted for 48 hours to induce insulin resistance in HepG2 cells. The cells were added with 0.75mL glycogen extract and ultrasonically disrupted at low temperature (power 200W, ultrasonic 3s, interval 10s, repeated 30 times), boiled in a boiling water bath for 20min, and then diluted to 5mL with distilled water after cooling, centrifuged at 8000×g, 25℃ for 10min, and the protein concentration was measured with a BCA protein concentration kit, and the intracellular glycogen content was determined by operating a glycogen detection kit. The results are as follows Fig.11 As shown, after intervention with different concentrations of SMDF-1, the glycogen accumulation of insulin-resistant cells increased to varying degrees.

[0089] (4) Effects of SMDF-1 on HK and PK activities in insulin-resistant HepG2 cells

[0090] HepG2 cells in the logarithmic growth phase were taken and the cell suspension obtained after trypsin digestion was diluted with complete medium to adjust the cell density to 1×10 5 / mL, inoculated in 96-well plates, 100μL per well, the cells were divided into normal group, insulin resistance group, positive control group, and drug group. Except for the normal group, 10 -8 mol / L insulin was used for 48h to induce insulin resistance in HepG2 cells. Hexokinase / pyruvate kinase extract was added at a ratio of 500:1 for the number of cells (cells): volume of extract (mL), and the cells were ultrasonically disrupted at low temperature (power 200W, ultrasonic 3s, interval 10s, repeated 30 times), centrifuged at 8000×g4℃ for 10min, and the supernatant was measured with a hexokinase (HK) / pyruvate kinase (PK) activity detection kit (Beijing Solebao). After high-concentration insulin stimulation, the HK and PK activities in the insulin resistance group cells decreased and were significantly lower than those in the normal group. After intervention with different concentrations of SMDF-1, the HK and PK activities of the model cells were significantly increased.

[0091] In summary, the test results of the embodiment show that mung bean sprout dietary fiber SMDF-1 can increase the glucose consumption, glycogen synthesis and HK and PK activities in HepG2 cells, thereby improving insulin resistance. Therefore, this component can become a potential functional food raw material for preventing and / or treating diabetes.

[0092] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents, without departing from the spirit and essence of the claims of the present invention; and these modifications or replacements are still within the scope defined by the claims of the present invention.

Claims

1. A mung bean sprout dietary fiber, characterized in that In terms of percentage by weight, the mung bean sprout dietary fiber comprises polysaccharides; The polysaccharide mainly consists of fucose, arabinose, galactose, glucose and mannose.

2. The mung bean sprout dietary fiber according to claim 1, characterized in that Its structural units are composed of →6)-α-D-Glcp-(1→, →5)-α-L-Araf-(1→ and →3,6)-α-D-Glcp-(1→, etc., which are interconnected to form the main chain, and the side chain is mainly composed of β-D-Manp-(1→ connected to the O-3 position of the sugar residue →3,6)-α-D-Glcp-(1→, etc.

3. The mung bean sprout dietary fiber according to claim 1, characterized in that The weight average molecular weight of the mung bean sprout dietary fiber is in the range of 100-200 kDa, preferably 150-180 kDa.

4. The mung bean sprout dietary fiber according to claim 1, characterized in that The molar ratio of fucose, arabinose, galactose, glucose and mannose is 10-15:2-5:25-30:5-10:45-55, preferably 12.28:2.60:27.59:7.06:50.

47.

5. The method for preparing mung bean sprout dietary fiber according to claims 1-4, characterized in that: The following steps are involved: a. Mung bean salt stress germination: Take fresh mung beans with full seeds, disinfect, stimulate, soak the seeds, place them in NaCl solution, and culture them in a constant temperature and humidity incubator away from light. b. Dietary fiber extraction: Take dried mung bean sprouts, crush them, remove fat and then perform enzymatic extraction. The obtained extract is concentrated, dialyzed and centrifuged to remove precipitation. The obtained supernatant is precipitated with alcohol and centrifuged. The obtained precipitate is washed and dried to obtain enzyme-extracted salt-stressed mung bean sprout dietary fiber. c. Dietary fiber purification: c1. The enzyme extract prepared in step b was dissolved in water for crude dietary fiber from salt-stressed mung bean sprouts, and then purified by an anion exchange column, eluted with water and 0.2 to 0.6 M NaCl solution, and the eluted fractions were collected to obtain salt-stressed mung bean sprouts dietary fiber MDF-0; c2. The dietary fiber MDF-0 prepared in step c1 was dissolved in about 80 to 100 times the weight of deionized water, purified by gel chromatography, eluted with deionized water, and the sulfated polysaccharide SMDF-1 component was collected and combined according to the elution curve to obtain the salt-stressed mung bean sprout dietary fiber SMDF-1.

6. The preparation method according to claim 5, characterized in that: The step a comprises: taking fresh mung beans with full seeds, disinfecting: soaking in 3% to 5% 84 disinfectant for half an hour, and rinsing with distilled water until there is no residue. Stimulating seeds: soaking mung beans in water at 40°C to 60°C for 2 minutes. Soaking seeds: soaking mung beans in water at 25°C to 30°C for 6 to 8 hours. The treated mung beans are evenly spread on a tray, and a NaCl solution with a concentration range of 0 to 0.15M is poured in. The culture solution is replaced every 12 hours, and the mung beans are placed in a constant temperature and humidity incubator at 25°C and 70% humidity to avoid light for culture. The step b comprises: taking dried mung bean sprouts, crushing them, adding 5 to 8 times the weight of anhydrous ethanol to remove small molecular substances such as lipids, adding distilled water to the defatted sample powder according to a solid-liquid ratio of 1:20 to 1:30, adjusting the pH value to 6 to 7, adding 1% to 2% alpha-amylase and saccharifying enzyme, extracting at 60°C for 3 to 5 hours, combining and concentrating the extracts, dialyzing, concentrating again, centrifuging to obtain a supernatant, adding anhydrous ethanol about 4 to 6 times the volume of the supernatant, centrifuging to obtain a precipitate, and vacuum freeze-drying to obtain enzyme-extracted salt-stressed mung bean sprout crude dietary fiber. The step c comprises: c1. taking the enzyme-extracted salt-stressed mung bean sprouts crude dietary fiber prepared in step b, adding about 10 times the weight of water to dissolve, separating through a DEAE cellulose DE-52 anion exchange column, eluting with deionized water, 0.2M, 0.4M and 0.6M NaCl solutions in sequence, detecting with sulfuric acid-phenol, collecting and merging the eluate of deionized water according to the elution curve, concentrating, centrifuging, taking the supernatant for dialysis, and freeze-drying to obtain the preliminarily purified dietary fiber MDF-0; c2. The dietary fiber MDF-0 prepared in step c1 was dissolved in about 100 times its weight of deionized water, purified by Sephadex G-100 polydextran gel column, eluted with deionized water, and detected by sulfuric acid-phenol method. The main component of dietary fiber SMDF-1 was collected and combined according to the elution curve, concentrated, dialyzed, and freeze-dried to obtain the salt-stressed mung bean sprout dietary fiber SMDF-1.

7. A salt-stressed mung bean sprout dietary fiber having the activity of improving insulin resistance obtained by the method according to any one of claims 1 to 4.

8. Use of the salt-stressed mung bean sprout dietary fiber with insulin resistance improving activity according to claim 7 in preparing functional foods or health products with the functions of improving glucose consumption and cell glycogen synthesis in insulin-resistant HepG2 cells and increasing the activities of hexokinase and pyruvate kinase.

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