White lotus non-starch polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the field of natural active substances, and particularly relates to white lotus non-starch polysaccharide as well as a preparation method and application thereof. The white lotus non-starch polysaccharide provided by the invention is a pyranose type polysaccharide with an alpha-configuration, and consists of (0.57% + / -0.47%) fucose, (11.75% + / -0.53%) rhamnose, (1.37% + / -0.73%) arabinose, (20.18% + / -0.19%) galactose, (49.44% + / -0.28%) glucose, (3.43% + / -0.35%) xylose, (2.93% + / -0.14%) mannose, (10.19% + / -0.12%) galacturonic acid and (0.13% + / -0.1%) glucuronic acid, and the molecular weight is Mw 145 KDa. The white lotus non-starch polysaccharide obtained by the invention has the potential of regulating the blood sugar level of patients with type II diabetes, and has important application value in the fields of development of foods, health care products and hypoglycemic drugs for the patients with diabetes.

Description

Technical Field

[0001] The invention belongs to the field of natural active substances, and particularly relates to a white lotus non-starch polysaccharide and a preparation method and application thereof. Background Art

[0002] While existing clinical hypoglycemic drugs such as metformin, nateglinide, and glucagonbay have demonstrated efficacy in treating type 2 diabetes, they are commonly associated with side effects such as abdominal distension, liver damage, and diarrhea, which can be inconvenient for patients. Furthermore, these drugs can develop varying degrees of drug resistance, and long-term use can lead to decreased efficacy, further limiting their clinical application. Therefore, the development of a harmless, highly effective, natural hypoglycemic agent has significant clinical and societal value.

[0003] White lotus, sweet and astringent in nature and flavor, enters the spleen, kidney, and heart meridians. It nourishes the heart and calms the mind, tonifies the spleen and intestines, strengthens the kidneys and nourishes the essence, and invigorates the spleen and removes dampness. It is a traditional Chinese medicine tonic and a valuable medicinal ingredient. Modern research has found that white lotus is rich in protein, carbohydrates, vitamins, and a variety of essential amino acids, and has immune-boosting, anti-aging, antioxidant, and anti-arrhythmic effects. Currently, research on white lotus has mostly focused on white lotus starch and some of its functional properties, with little research on the processing and product development of its other active ingredients. However, white lotus starch has a glycemic index greater than 90, making it a high-glycemic index food, which limits its use in the diets of diabetic patients. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a white lotus non-starch polysaccharide and its preparation method and application. The white lotus non-starch polysaccharide provided by the present invention is extracted from white lotus and is a natural blood sugar-lowering ingredient with no side effects and high efficiency, providing a new treatment option for diabetic patients.

[0005] In the first aspect, the present invention provides a white lotus non-starch polysaccharide, which is a pyranose polysaccharide with an α-configuration, composed of (0.57%±0.47%) fucose, (11.75%±0.53%) rhamnose, (1.37%±0.73%) arabinose, (20.18%±0.19%) galactose, (49.44%±0.28%) glucose, (3.43%±0.35%) xylose, (2.93%±0.14%) mannose, (10.19%±0.12%) galacturonic acid and (0.13%±0.1%) glucuronic acid, with a molecular weight of Mw 145 KDa.

[0006] The white lotus non-starch polysaccharide provided by the present invention is obtained by separation and purification from white lotus. Its structural characteristics are as follows: a linear main chain is composed of glucose (Glc) units connected by (→4)-α-Glc-(1→), similar to the structure of starch or glucan; the side chain part is composed of galactose (Gal) and rhamnose (Rha) connected by (→3)-β-Gal-(1→) or (→2)-α-Rha-(1→) to form branches; the polysaccharide component contains 10.19% galacturonic acid (GalA), indicating the presence of (→4)-α-GalA-(1→) linkages in the main chain or side chains. This structural feature is consistent with the typical component units of pectin polysaccharides, suggesting that the polysaccharide may have the composite structural characteristics of both glucan and pectin polysaccharides.

[0007] The white lotus non-starch polysaccharide provided by the present invention exhibited significant hypoglycemic effects in both in vitro and in vivo hypoglycemic studies. In vitro enzyme inhibition experiments demonstrated that the polysaccharide exhibited significant inhibitory effects on α-amylase and α-glucosidase activities (inhibition rates of 62.56±2.01% and 87.06±0.24%, respectively), suggesting that it exerts its hypoglycemic effect by delaying carbohydrate hydrolysis. In diabetic mice induced by a high-fat, high-sugar diet combined with a low-dose streptozotocin (STZ), daily oral administration of 200 mg / kg for five weeks demonstrated systemic metabolic improvements: compared to the model group, body weight increased by 33.01%, blood glucose decreased by 115.38%, and the area under the curve (AUC) of the oral insulin tolerance test (ITT) decreased by 26.43%. Furthermore, the polysaccharide significantly reduced the levels of glycosylated serum proteins and serum insulin in mice, decreased the HOMA-IR index, and improved insulin sensitivity. Analysis of intestinal unsaturated fatty acids and microbial composition revealed that diabetic mice treated with non-starch polysaccharides from white lotus exhibited altered intestinal microbial composition and increased unsaturated fatty acid content. In vivo experiments suggest that non-starch polysaccharides from white lotus may regulate glucose homeostasis through a multi-target mechanism.

[0008] In a second aspect, the present invention also provides a method for preparing the white lotus non-starch polysaccharide, the preparation method comprising the following steps: Step 1: Wash and core the white lotus, dry and grind it to obtain coarse white lotus powder; Step 2: adding water to the crude white lotus powder obtained in step 1 and mixing to obtain a suspension; subjecting the suspension to pressurized water-eddy current pulse electric field combined extraction, solid-liquid separation, and collecting the supernatant to obtain a white lotus extract; Step 3: adding immobilized enzyme microspheres to the white lotus extract obtained in step 2 at a ratio of 40-60 g immobilized enzyme microspheres per 100 g crude white lotus powder, performing enzymatic hydrolysis at 50-60° C. until the iodine solution color reaction of the white lotus extract turns colorless or light yellow, thereby obtaining an enzymatic hydrolyzate; performing solid-liquid separation on the enzymatic hydrolyzate, taking the supernatant for alcohol precipitation, and collecting the precipitate; removing residual ethanol from the precipitate, and drying to obtain crude white lotus polysaccharide; the immobilized enzyme microspheres are enzyme-biomass-based gel complexes, and the enzymes are one or more enzymes capable of degrading white lotus starch or cellulose; Step 4: dissolving the crude polysaccharide of white lotus in water to prepare a crude polysaccharide solution; subjecting the crude polysaccharide solution to column chromatography, wherein the column chromatography uses a D101 macroporous resin column as a stationary phase and distilled water as an elution solvent; when the molish reaction of the eluate is negative, the elution is stopped, and the collected eluate is concentrated and dried to obtain the crude non-starch polysaccharide of white lotus; Step 5: dissolving the crude non-starch polysaccharide of white lotus in water to prepare a non-starch polysaccharide solution, filtering the obtained filtrate and performing gel filtration chromatography, wherein the gel filtration chromatography adopts a Sephacryl S-400 HR gel filtration column, the elution solvent is 0.1 M sodium chloride solution, and the eluate corresponding to the polysaccharide peak in the absorbance curve at 490 nm is collected; the eluate is dialyzed through an 8000-12000 Da dialysis bag, and the dialyzed retentate is dried to obtain the non-starch polysaccharide of white lotus.

[0009] In step 1, the dried white lotus powder is helpful for subsequent extraction. Optionally, the particle size of the coarse white lotus powder is ≤0.5 mm.

[0010] The drying method in steps 1, 3, 4 and 5 can be direct drying, concentration followed by drying, or reduced pressure vacuum drying, etc., and the drying can be performed to a constant weight.

[0011] The endpoint of the enzymatic hydrolysis in step 3 is a negative iodine solution color reaction of the white lotus extract, that is, no starch in the white lotus extract can be detected by the iodine solution color reaction, and this is used as a standard to control the enzymatic hydrolysis time. The present invention uses the iodine solution color reaction to indicate the starch content in the white lotus extract corresponding to the enzymatic hydrolysis endpoint, but this does not mean that the iodine solution color reaction must be performed in this step, nor does it mean that the method for detecting starch content is limited to the iodine solution color reaction.

[0012] In step 3, solid-liquid separation can be performed by filtration or centrifugation, and the corresponding filtrate or supernatant can be collected. In the alcohol precipitation process, ethanol is slowly added to the filtrate or supernatant, and the mixture is allowed to stand until the precipitate no longer increases, generally for 8-12 hours.

[0013] During column chromatography in step 4, the elution endpoint is when the eluate shows a negative molish reaction, i.e., when no polysaccharide can be detected in the eluate by the molish reaction. This endpoint is then reached, and the elution time is controlled based on this criterion. The present invention uses the molish reaction to indicate the polysaccharide content in the eluate at the elution endpoint, but this does not necessarily mean that a molish reaction is required in this step, nor does it mean that the method for detecting polysaccharides in the eluate is limited to the molish reaction.

[0014] The white lotus polysaccharide extracted by the current polysaccharide extraction technology has the problem of low content, and the high content of starch not only affects its purity and quality, but also limits its application in blood sugar regulation.

[0015] The preparation method of white lotus non-starch polysaccharides provided by the present invention significantly increases the content of white lotus non-starch polysaccharides and reduces the content of impurities such as starch and protein. The polysaccharide content in the obtained extract is about 80%. This method first subjects crude white lotus powder to high-temperature and high-pressure, high-frequency eddy current and pulsed electric field multi-physical field synergistic extraction. This method can enhance solvent permeability, improve mass transfer efficiency, increase cell wall porosity, increase solid-liquid contact area and promote polysaccharide dissolution; then, enzymatic hydrolysis is performed using immobilized enzymes to improve the enzymatic hydrolysis efficiency of starch. Afterwards, crude white lotus polysaccharides are obtained by alcohol precipitation; and the crude white lotus polysaccharides are further separated and purified to obtain a high-content target polysaccharide. This method achieves high-content extraction of white lotus non-starch polysaccharides by optimizing the extraction and purification processes.

[0016] Preferably, the crude white lotus powder and water in step 2 are mixed at a material-liquid ratio of 1:15-20 (g / mL), and soaked until the soluble components are fully dissolved to obtain a suspension; the suspension is treated at 110°C, 0.4 MPa, and 200 rpm for 30-40 min; and then a pulsed electric field of 15 kV / cm and an eddy current of 30 kHz are applied for 10-20 min.

[0017] Preferably, the method further includes the preparation of the immobilized enzyme microspheres described in step 3; the preparation method includes: dissolving the enzyme in phosphate buffer to prepare an enzyme solution; adding biomass-based gel microspheres with a diameter of 2-3 mm to the enzyme solution, shaking and adsorbing the biomass-based gel microspheres sufficiently, and then adding a cross-linking agent to form a covalently bond-stabilized enzyme-biomass-based gel complex; after cross-linking, collecting the immobilized enzyme microspheres by centrifugation, removing unbound free enzyme and residual cross-linking agent, and obtaining immobilized enzyme microspheres.

[0018] More preferably, the enzyme in step 3 is a combination of amylase and cellulase; the mass ratio of amylase to cellulase in the enzyme solution is 3:1; the concentration of the enzyme in the enzyme solution is 10-20 mg / mL; and biomass-based gel microspheres are added to the enzyme solution at a ratio of 2 mL enzyme solution / g.

[0019] More preferably, the biomass-based gel microspheres are sodium alginate-based gel microspheres; and the cross-linking agent is genipin.

[0020] More preferably, the oscillating adsorption is performed at 150 rpm for 8 hours.

[0021] More preferably, the cross-linking conditions are 37° C., protected from light, 50 rpm, and time for 24 h.

[0022] Preferably, the immobilized enzyme microspheres are added to the white lotus extract in step 3 at a ratio of 60 g immobilized enzyme microspheres per 100 g crude white lotus powder.

[0023] Preferably, the alcohol precipitation comprises: adding ethanol to the supernatant to make the mass percentage of ethanol in the solution reach 70%-80%, stirring sufficiently, and standing until the weight of the precipitate no longer increases.

[0024] Preferably, the concentration of the crude polysaccharide solution in step 4 is 10-20 mg / mL, and elution is performed more than 8 hours after adsorption.

[0025] Preferably, in step 5, the concentration of the non-starch polysaccharide solution is 20 mg / mL, the flow rate of the elution solvent is controlled to be 0.8 mL / min, and the eluate with an elution time of 11.8-15.3 min is collected.

[0026] In a third aspect, the present invention further provides the use of the white lotus non-starch polysaccharide and the white lotus non-starch polysaccharide prepared according to the above preparation method in food, health food and medicine.

[0027] Preferably, the invention is used in health foods that help maintain blood sugar health or help regulate intestinal flora; Application in medicines for lowering blood sugar, alleviating insulin resistance or reducing glycosylated serum protein levels; Application in medicines for regulating intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is a high-performance gel permeation chromatogram of the white lotus non-starch polysaccharide provided by the present invention; Figure 2 The monosaccharide composition of the white lotus non-starch polysaccharide provided by the present invention; Figure 3 This is the infrared spectrum of the white lotus non-starch polysaccharide provided by the present invention; Figure 4 The non-starch polysaccharide of white lotus provided by the present invention has an inhibitory effect on α-amylase; Figure 5 The non-starch polysaccharide of white lotus provided by the present invention has an inhibitory effect on α-glucosidase; Figure 6 The fasting body weight changes of mice in Example 4; Figure 7 This is the change in fasting blood glucose in mice in Example 4; Figure 8 This is the fasting oral glucose tolerance test of mice in Example 4; Figure 9 is the insulin tolerance of mice in Example 4; Figure 10 is the serum insulin concentration and insulin resistance index of the mice in Example 4; Figure 11 is the glycosylated serum protein content of mice in Example 4; Figure 12 Comparison of the overall intestinal flora levels of mice in each group in Example 4; Figure 13 Comparison of the levels of six major intestinal flora in each group of mice in Example 4; Figure 14 is the content of short-chain fatty acids in the cecal contents of each group of mice in Example 4. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] White lotus, Guangchang County Junzhi Food Co., Ltd.; mice, Jiangsu Jicui Pharmaceutical Co., Ltd.

[0032] Amylase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., model A109181; cellulase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., model C757787.

[0033] Example 1 This embodiment provides a white lotus non-starch polysaccharide extract, and the specific preparation method is as follows: Step 1: Take fresh white lotus, clean it, remove the core, and dry it until the mass is constant and the moisture content is about 4%. Crush it with a powder grinder and pass it through a 60-mesh sieve to obtain coarse white lotus powder.

[0034] Step 2: Crude white lotus powder was mixed with deionized water at a solid-liquid ratio of 1:15 (g / mL) and soaked for 30 minutes until the soluble components were fully dissolved to obtain a suspension. The suspension was then transferred to a stainless steel high-pressure reactor and dynamically extracted at 110°C, 0.4 MPa pressure, and a rate of 200 rpm (counter-clockwise / counter-clockwise switching every 5 minutes) for 30 minutes. The suspension was then connected to an eddy current-pulsed electric field system with titanium alloy electrodes and synergistically treated at a field strength of 15 kV / cm (pulse width 10 μs, frequency 100 Hz) and a 30 kHz eddy current frequency (power density 50 W / L) for 20 minutes. After centrifugation, the supernatant was collected and concentrated to 1 / 4 of the original supernatant volume to obtain the white lotus extract.

[0035] Step 3: Preparation of sodium alginate-based gel microspheres: Weigh 1% sodium alginate, add it to deionized water, and dissolve it fully under stirring conditions at a stirring speed of 300 r / min. The temperature is maintained at 60°C to accelerate the dissolution process and obtain a uniform sodium alginate solution; weigh sodium chloride, dissolve it in deionized water to prepare a 0.5 mol / L sodium chloride solution, and slowly add the sodium chloride solution to the sodium alginate solution while stirring at a stirring speed of 100 r / min to fully mix the two; the mixed solution is dripped into 10 times the volume of 0.1 M calcium chloride solution through a syringe pump and allowed to stand at room temperature for 30 min to form gel microspheres with a diameter of 2-3 mm. The solution is washed with deionized water three times to remove residual Ca 2+ Preparation of enzyme solution: Take a certain amount of amylase and cellulase at a mass ratio of 3:1 and dissolve them in phosphate buffered saline (PBS) at pH 7.4 to prepare an enzyme solution with a concentration of 10 mg / mL. Enzyme immobilization: Add 2 mL of enzyme solution / g to sodium alginate-based gel microspheres. Oscillating and adsorbing at 4°C and 150 rpm for 8 hours, add genipin solution (final concentration 0.5% w / v, solvent 50% ethanol), crosslink at 37°C in the dark for 24 hours (rotation speed 50 rpm), and wash with PBS three times to remove uncrosslinked enzyme and genipin, completing enzyme immobilization and obtaining immobilized enzyme microspheres.

[0036] Step 4: Add immobilized enzyme microspheres to the white lotus extract obtained in step 2 at a ratio of 40 g immobilized enzyme microspheres per 100 g crude white lotus powder, perform enzymolysis at a temperature of 50°C and 250 rpm for 2 h to obtain an enzymatic hydrolyzate; centrifuge the enzymatic hydrolyzate at a speed of 1200 rpm for 10 minutes, and collect the supernatant; add ethanol to the supernatant to make the ethanol mass percentage in the solution reach 70%, stir for 1 h, let it stand overnight, centrifuge, collect the precipitate, re-dissolve the precipitate with distilled water, stir until completely dissolved, remove the ethanol residue using a rotary evaporator (temperature of 60-65°C, pressure of 60-65 mbar, shaft speed of 60-65 rpm), concentrate, and dry in an oven to obtain white lotus crude polysaccharide.

[0037] Step 5: Dissolve the crude polysaccharide from white lotus in water to prepare a 10 mg / mL crude polysaccharide solution, apply it to a D101 macroporous resin column (2.5×50 cm) with a sample volume of 15 mL. After adsorption for 12 h, elute with distilled water and collect the eluate. When the molish reaction of the eluate is negative, stop elution; concentrate and dry the eluate to obtain the crude non-starch polysaccharide from white lotus.

[0038] Step 6: The crude non-starch polysaccharide of white lotus was dissolved in water to prepare a 20 mg / mL non-starch polysaccharide solution. The filtrate obtained after filtering with a 0.45 μm filter membrane was subjected to gel filtration chromatography. The gel filtration chromatography used a Sephacryl S-400 HR gel filtration column, the sample volume was 10 mL, the elution solvent was 0.1 M sodium chloride solution, the flow rate was 0.8 mL / min, and the eluate with an elution time between 11.8 and 15.3 min was collected; the collected eluate was dialyzed with a 12000 Da dialysis bag, and the dialyzed retentate was concentrated on a reduced pressure vacuum rotary evaporator and then freeze-dried with a vacuum freeze dryer to obtain the white lotus non-starch polysaccharide extract 1.

[0039] Example 2 This embodiment provides a white lotus non-starch polysaccharide extract, and the specific preparation method is as follows: Step 1: Take fresh white lotus, clean it, remove the core, and dry it until the mass is constant and the moisture content is about 4%. Crush it with a powder grinder and pass it through a 60-mesh sieve to obtain coarse white lotus powder.

[0040] Step 2: Crude white lotus powder was mixed with deionized water at a solid-liquid ratio of 1:17 (g / mL) and soaked for 40 minutes until the soluble components were fully dissolved to obtain a suspension. The suspension was then transferred to a stainless steel high-pressure reactor and dynamically extracted at 110°C, 0.4 MPa pressure, and a rate of 200 rpm (counterclockwise / counterclockwise switching every 5 minutes) for 35 minutes. The suspension was then connected to an eddy current-pulsed electric field system with titanium alloy electrodes and synergistically treated at a field strength of 15 kV / cm (pulse width 10 μs, frequency 100 Hz) and a 30 kHz eddy current frequency (power density 50 W / L) for 15 minutes. After centrifugation, the supernatant was collected and concentrated to 1 / 4 of the original supernatant volume to obtain the white lotus extract.

[0041] Step 3: Preparation of sodium alginate-based gel microspheres: Weigh 1.5% sodium alginate and add it to deionized water. Dissolve it fully under stirring at a speed of 400 r / min and maintain the temperature at 50°C to accelerate the dissolution process and obtain a uniform sodium alginate solution. Weigh sodium chloride and dissolve it in deionized water to prepare a 0.5 mol / L sodium chloride solution. Slowly add the sodium chloride solution to the sodium alginate solution while stirring at a speed of 100 r / min to fully mix the two. The mixed solution is dripped into 10 times the volume of 0.1 M calcium chloride solution through a syringe pump and allowed to stand at room temperature for 30 minutes to form gel microspheres with a diameter of 2-3 mm. Wash with deionized water three times to remove residual Ca 2+ Preparation of enzyme solution: Take a certain amount of amylase and cellulase at a mass ratio of 3:1 and dissolve them in phosphate buffered saline (PBS) at pH 7.4 to prepare an enzyme solution with a concentration of 15 mg / mL. Enzyme immobilization: Add 2 mL of enzyme solution / g to sodium alginate-based gel microspheres. Oscillate and adsorb at 4°C and 150 rpm for 8 hours. Add genipin solution (final concentration 0.5% w / v, solvent 50% ethanol), crosslink at 37°C in the dark for 24 hours (rotation speed 50 rpm), and wash with PBS three times to remove uncrosslinked enzyme and genipin. This completes enzyme immobilization and produces immobilized enzyme microspheres.

[0042] Step 4: Add immobilized enzyme microspheres to the white lotus extract obtained in step 2 at a ratio of 50 g immobilized enzyme microspheres per 100 g crude white lotus powder, perform enzymolysis at a temperature of 55°C and 250 rpm for 1.5 h to obtain an enzymatic hydrolyzate; centrifuge the enzymatic hydrolyzate at a speed of 1200 rpm for 10 minutes, and collect the supernatant; add ethanol to the supernatant to make the ethanol mass percentage in the solution reach 75%, stir for 1 h, let it stand overnight, centrifuge, collect the precipitate, redissolve the precipitate with distilled water, stir until completely dissolved, remove the ethanol residue using a rotary evaporator (temperature of 60-65°C, pressure of 60-65 mbar, shaft speed of 60-65 rpm), concentrate, and dry in an oven to obtain white lotus crude polysaccharide.

[0043] Step 5: Dissolve the crude polysaccharide from white lotus in water to prepare a 15 mg / mL crude polysaccharide solution, apply it to a D101 macroporous resin column (2.5×50 cm) with a sample volume of 15 mL. After adsorption for 12 h, elute with distilled water and collect the eluate. When the molish reaction of the eluate is negative, stop elution; concentrate and dry the eluate to obtain the crude non-starch polysaccharide from white lotus.

[0044] Step 6: The crude non-starch polysaccharide of white lotus was dissolved in water to prepare a 20 mg / mL non-starch polysaccharide solution. The filtrate obtained after filtering with a 0.45 μm filter membrane was subjected to gel filtration chromatography. The gel filtration chromatography used a Sephacryl S-400 HR gel filtration column, the sample volume was 8 mL, the elution solvent was 0.1 M sodium chloride solution, the flow rate was 0.8 mL / min, and the eluate with an elution time between 11.8-15.3 min was collected; the collected eluate was dialyzed with a 12000 Da dialysis bag, and the dialyzed retentate was concentrated on a reduced pressure vacuum rotary evaporator and then freeze-dried with a vacuum freeze dryer to obtain the white lotus non-starch polysaccharide extract 2.

[0045] Example 3 This embodiment provides a white lotus non-starch polysaccharide extract, and the specific preparation method is as follows: Step 1: Take fresh white lotus, clean it, remove the core, and dry it until the mass is constant and the moisture content is about 4%. Crush it with a powder grinder and pass it through a 60-mesh sieve to obtain coarse white lotus powder.

[0046] Step 2: Crude white lotus powder was mixed with deionized water at a solid-liquid ratio of 1:20 (g / mL) and soaked for 50 minutes until the soluble components were fully dissolved to obtain a suspension. The suspension was then transferred to a stainless steel high-pressure reactor and dynamically extracted at 110°C, 0.4 MPa pressure, and a rate of 200 rpm (counter-clockwise / counter-clockwise switching every 5 minutes) for 40 minutes. The suspension was then connected to an eddy current-pulsed electric field system with titanium alloy electrodes and synergistically treated at a field strength of 15 kV / cm (pulse width 10 μs, frequency 100 Hz) and a 30 kHz eddy current frequency (power density 50 W / L) for 20 minutes. After centrifugation, the supernatant was collected and concentrated to 1 / 4 of the original supernatant volume to obtain the white lotus extract.

[0047] Step 3: Preparation of sodium alginate-based gel microspheres: Weigh 2% sodium alginate, add it to deionized water, and fully dissolve it under stirring conditions at a stirring speed of 500 r / min. The temperature is maintained at 60°C to accelerate the dissolution process and obtain a uniform sodium alginate solution; weigh sodium chloride, dissolve it in deionized water to prepare a 0.5 mol / L sodium chloride solution, and slowly add the sodium chloride solution to the sodium alginate solution while stirring at a stirring speed of 100 r / min to fully mix the two; the mixed solution is dripped into 10 times the volume of 0.1 M calcium chloride solution through a syringe pump and allowed to stand at room temperature for 30 min to form gel microspheres with a diameter of 2-3 mm. The solution is washed with deionized water three times to remove residual Ca 2+ Preparation of enzyme solution: Take a certain amount of amylase and cellulase at a mass ratio of 3:1 and dissolve them in phosphate buffered saline (PBS) at pH 7.4 to prepare an enzyme solution with a concentration of 20 mg / mL. Enzyme immobilization: Add sodium alginate-based gel microspheres at a ratio of 2 mL of enzyme solution / g. Oscillate and adsorb at 4°C and 150 rpm for 8 hours. Add genipin solution (final concentration 0.5% w / v, solvent 50% ethanol), crosslink at 37°C in the dark for 24 hours (rotation speed 50 rpm), and wash with PBS three times to remove uncrosslinked enzyme and genipin. This completes enzyme immobilization and produces immobilized enzyme microspheres.

[0048] Step 4: Add immobilized enzyme microspheres to the white lotus extract obtained in step 2 at a ratio of 60 g immobilized enzyme microspheres per 100 g crude white lotus powder, perform enzymolysis at a temperature of 60°C and 250 rpm for 1.5 h to obtain an enzymatic hydrolyzate; centrifuge the enzymatic hydrolyzate at a speed of 1200 rpm for 10 minutes, and collect the supernatant; add ethanol to the supernatant to make the ethanol mass percentage in the solution reach 80%, stir for 1 h, let it stand overnight, centrifuge, collect the precipitate, redissolve the precipitate with distilled water, stir until completely dissolved, remove the ethanol residue using a rotary evaporator (temperature of 60-65°C, pressure of 60-65 mbar, shaft speed of 60-65 rpm), concentrate, and dry in an oven to obtain white lotus crude polysaccharide.

[0049] Step 5: Dissolve the crude polysaccharide from white lotus in water to prepare a 10 mg / mL crude polysaccharide solution, apply it to a D101 macroporous resin column (2.5×50 cm) with a sample volume of 15 mL. After adsorption for 12 h, elute with distilled water and collect the eluate. When the molish reaction of the eluate is negative, stop elution; concentrate and dry the eluate to obtain the crude non-starch polysaccharide from white lotus.

[0050] Step 6: The crude non-starch polysaccharide of white lotus was dissolved in water to prepare a 20 mg / mL non-starch polysaccharide solution. The filtrate obtained after filtering with a 0.45 μm filter membrane was subjected to gel filtration chromatography. The gel filtration chromatography used a Sephacryl S-400 HR gel filtration column, the sample volume was 10 mL, the elution solvent was 0.1 M sodium chloride solution, the flow rate was 0.8 mL / min, and the eluate with an elution time between 11.8 and 15.3 min was collected; the collected eluate was dialyzed with a 12000 Da dialysis bag, and the dialyzed retentate was concentrated on a reduced pressure vacuum rotary evaporator and then freeze-dried with a vacuum freeze dryer to obtain the white lotus non-starch polysaccharide extract 3.

[0051] Comparative Example 1 This comparative example provides a white lotus non-starch polysaccharide extract. Its specific preparation method is essentially the same as that of Example 3, except that, in step 2, the extraction method is hot water extraction. White lotus powder and deionized water are mixed at a material-to-liquid ratio of 1:20 (g / mL) to obtain a suspension. The suspension is then incubated in a 90°C water bath for 2 hours, centrifuged, and the supernatant is collected. The separated precipitate is subjected to a second extraction, using the same amount of water and time as the first extraction. The supernatant is then collected after centrifugation, and the two supernatants are combined and concentrated to 1 / 8 the volume of the original supernatant to obtain the white lotus extract. The remaining steps are the same as those of Example 3, ultimately yielding white lotus non-starch polysaccharide extract 4.

[0052] Comparative Example 2 This embodiment provides a white lotus non-starch polysaccharide extract, and the specific preparation method is as follows: Step 1: Take fresh white lotus, clean it, remove the core, and dry it until the mass is constant and the moisture content is about 4%. Crush it with a powder grinder and pass it through a 60-mesh sieve to obtain coarse white lotus powder.

[0053] Step 2: Crude white lotus powder was mixed with deionized water at a solid-liquid ratio of 1:20 (g / mL) and soaked for 30 minutes until the soluble components were fully dissolved to obtain a suspension. The suspension was then transferred to a stainless steel high-pressure reactor and dynamically extracted at 110°C, 0.4 MPa pressure, and a rate of 200 rpm (counterclockwise / counterclockwise switching every 5 minutes) for 40 minutes. The suspension was then connected to an eddy current-pulsed electric field system with titanium alloy electrodes and synergistically treated at a field strength of 15 kV / cm (pulse width 10 μs, frequency 100 Hz) and a 30 kHz eddy current frequency (power density 50 W / L) for 20 minutes. After centrifugation, the supernatant was collected and concentrated to 1 / 4 of the original supernatant volume to obtain the white lotus extract.

[0054] Step 3: Add 1.5 g of amylase and cellulase (amylase: cellulase = 3:1) per 100 g of crude white lotus powder to the white lotus extract obtained in step 2, and perform enzymolysis at 60°C and 250 rpm for 1.5 h to obtain an enzymatic solution; after the enzymatic solution is inactivated at 100°C for 20 minutes, it is quickly cooled in an ice bath and centrifuged at 12,000 rpm and 4°C for 15 minutes using a refrigerated centrifuge to collect the supernatant; add ethanol to the supernatant to make the ethanol content in the solution reach 80%, stir for 1 h, let it stand overnight, centrifuge, collect the precipitate, re-dissolve the precipitate with distilled water, stir until completely dissolved, remove the ethanol residue using a rotary evaporator (temperature 60-65°C, pressure 60-65 mbar, shaft speed 60-65 rpm), concentrate, and dry in an oven to obtain white lotus crude polysaccharide.

[0055] Step 4: Dissolve the crude polysaccharide from white lotus in water to prepare a 10 mg / mL crude polysaccharide solution, apply it to a D101 macroporous resin column (2.5×50 cm) with a sample volume of 15 mL. After adsorption for 12 h, elute with distilled water and collect the eluate. When the molish reaction of the eluate is negative, stop elution; concentrate and dry the eluate to obtain the crude non-starch polysaccharide from white lotus.

[0056] Step 5: The crude non-starch polysaccharide of white lotus was dissolved in water to prepare a 20 mg / mL non-starch polysaccharide solution. The filtrate obtained after filtering with a 0.45 μm filter membrane was subjected to gel filtration chromatography. The gel filtration chromatography used a Sephacryl S-400 HR gel filtration column, the sample volume was 10 mL, the elution solvent was 0.1 M sodium chloride solution, the flow rate was 0.8 mL / min, and the eluate with an elution time between 11.8-15.3 min was collected; the collected eluate was dialyzed with a 12000 Da dialysis bag, and the dialyzed retentate was concentrated on a reduced pressure vacuum rotary evaporator and then freeze-dried with a vacuum freeze dryer to obtain the white lotus non-starch polysaccharide extract 5.

[0057] Verification Example 1 The polysaccharide, protein, and starch contents of the white lotus non-starch polysaccharide extracts of Examples 1-3 and Comparative Examples 1-2 were measured, and the results are shown in Table 1. The neutral sugar content was determined using the phenol-sulfuric acid method, the acidic sugar content was determined using the carbazole-ethanol method, the starch content was determined using a starch content assay kit (Shanghai Xinfan Biotechnology Co., Ltd.), and the protein content was determined using the Coomassie Brilliant Blue method.

[0058] Table 1 Content of components in non-starch polysaccharides of white lotus

[0059] The white lotus non-starch polysaccharide extracted by the preparation method provided by the invention has a non-starch polysaccharide content of about 80% and a small content of impurities such as starch and protein.

[0060] In contrast, the non-starch polysaccharide content in the polysaccharide extracts prepared in Comparative Example 1 and Comparative Example 2 decreased significantly, indicating that the combined extraction method of pressurized water (PHW)-eddy current pulsed electric field and the enzymatic hydrolysis method of immobilized enzymes can promote the release of sugars and efficiently degrade starch, thereby achieving the removal of starch and increasing the non-starch polysaccharide content in the extract.

[0061] Verification Example 1 Characterization of White Lotus Non-Starch Polysaccharides The molecular weight of polysaccharide was determined by high performance gel permeation chromatography (HPLC). Figure 1 shown.

[0062] from Figure 1 It can be seen that the chromatographic peak of the white lotus non-starch polysaccharide LSPS is a single peak and is normally distributed, indicating that the LSPS obtained by gel column chromatography separation and purification is a relatively uniform component with a weight average molecular weight Mw of 145 KDa.

[0063] Determination of Monosaccharide Composition The HPAEC-PAD method was used to quantitatively analyze the proportion of each monosaccharide in the white lotus non-starch polysaccharide (LSPS, provided in Example 3). The results are shown in Tables 2 and Figure 2 .

[0064] 5 mg of white lotus non-starch polysaccharide was weighed into a test tube. 0.5 mL of 12 mol / L concentrated sulfuric acid was added and stirred for 30 minutes under an ice bath. 2.5 mL of ultrapure water was then added and stirred for 1 minute. The mixture was then transferred to a 120°C oil bath and stirred for 1 hour. The volume was then diluted to a 50 mL volumetric flask, filtered through a 0.22 μm water filter, and analyzed on a Dionex ICS 6000 ion chromatograph. Simultaneously, monosaccharide standards (fucose, rhamnose, galactose, arabinose, xylose, glucose, mannose, fructose, ribose, galacturonic acid, and glucuronic acid) at varying concentrations were prepared for chromatographic analysis to determine the types and contents of monosaccharides in the samples. All experiments were repeated three times.

[0065] Table 2 Monosaccharide composition and ratio of white lotus non-starch polysaccharides

[0066] Analysis of the monosaccharide composition of the lotus non-starch polysaccharide LSPS shows that it is primarily composed of three monosaccharides: glucose (Glc), galactose (Gal), and rhamnose (Rha), accounting for approximately 81% of the polysaccharide content. The molar ratio of these three monosaccharides is Glc:Gal:Rha = 4.2:1.7:1. LSPS also contains smaller amounts of monosaccharides such as arabinose (Ara), xylose (Xyl), and mannose (Man), as well as the uronic acid derivatives galacturonic acid (GalA) and glucuronic acid (GlcA).

[0067] Infrared spectral analysis The white lotus non-starch polysaccharide (LSPS, provided in Example 3) was subjected to infrared spectral analysis.

[0068] 1 mg of LSPS was mixed with 150 mg of potassium bromide powder, ground thoroughly, and then pressed into transparent sheets. The samples were analyzed by Fourier transform infrared spectroscopy (FT-IR) at 400-4000 cm -1 Scanning within a range of 4 cm -1 The infrared spectrum of white lotus non-starch polysaccharide LSPS is as follows: Figure 3 shown.

[0069] Depend on Figure 3 It can be seen that the infrared spectrum of LSPS is at 3409.58 cm -1 stretching vibration of the OH bond at 2929.39 cm -1 The stretching vibration of the CH bond at 597.83 cm and the absorption peak at 1633.44 cm are characteristic peaks of polysaccharides; -1The absorption peak at 1074.17 cm is derived from the angular vibration of β-type CH bond; -1 and 1073.57 cm -1 The characteristic peak at 1253.52 cm corresponds to the pyranose ring structure connected by oxygen bridges at C1 and C5, and the peak at 1253.52 cm -1 The symmetrical stretching vibration of the C-O-C bridge bond in the pyran ring indicates that the polysaccharide has a stable pyran ring configuration; 1421.30 cm -1 The peak at is attributed to the symmetrical stretching vibration of C=O in uronic acid, indicating the presence of uronic acid in the sugar chain.

[0070] The above results indicate that the white lotus non-starch polysaccharide provided by the present invention is a pyranose polysaccharide with an α-configuration. Its linear backbone is primarily composed of glucose (Glc), linked by (→4)-α-Glc-(1→), similar to the structure of starch or glucan. The side chains are primarily composed of galactose (Gal) and rhamnose (Rha), connected by (→3)-β-Gal-(1→) or (→2)-α-Rha-(1→), forming branches. Galacturonic acid (GalA) is also present in the backbone or side chains, linked by (→4)-α-GalA-(1→), similar to the structure of pectin polysaccharides.

[0071] Example 4 Analysis of the Hypoglycemic Activity Potential of White Lotus Non-Starch Polysaccharide (LSPS, provided in Example 3) 1. Inhibition of α-amylase activity in vitro by non-starch polysaccharides from white lotus (1) Solution preparation: Take 0.5 g of starch and add it to an appropriate amount of 0.1 mol / L phosphate buffer at pH 6.8. After thorough oscillation and mixing, place the solution in a boiling water bath and heat until the starch is completely dissolved. After the solution cools to room temperature, dilute it to 50 mL with phosphate buffer to obtain a 1% starch solution for later use. Accurately weigh α-amylase powder, dissolve the α-amylase powder with phosphate buffer, and dilute it to a concentration of 0.1 U / mL to obtain a 0.1 U / mL α-amylase solution for later use. Take an appropriate amount of white lotus non-starch polysaccharide sample, completely dissolve it with distilled water, and after dilution, prepare a series of LSPS solutions with concentrations of 0.1, 0.25, 0.5, 1, 2, 4, and 8 mg / mL, respectively, for later use. Weigh acarbose, dissolve it with distilled water, and dilute it to prepare a series of positive control solutions with concentrations of 0.1, 0.25, 0.5, 1, 2, 4, and 8 mg / mL, for later use.

[0072] (2) Detection: The inhibition rate of LSPS on α-amylase activity was determined by the DNS method, with acarbose as a positive control. 100 μL LSPS solution and 300 μL α-amylase solution were placed in a test tube. After incubating in a 37°C water bath for 5 minutes, 400 μL 1% starch solution was added. After mixing evenly and incubating in a 37°C water bath for 5 minutes, 200 μL DNS reagent was added to terminate the reaction. The tube was boiled at 100°C for 15 minutes, cooled, and then the color was developed after incubating in a boiling water bath for 5 minutes. The tube was taken out and cooled to room temperature. The absorbance A was measured at a wavelength of 540 nm. i , repeated 3 times, the inhibition rate was calculated according to formula (1), and the results are shown in Figure 4 .

[0073] Inhibition rate (%) = [1-(A1-A2) / (A0-A3)]×100 (1) Wherein, A0: replace the test solution with an equal volume of distilled water; A2: replace the α-amylase solution with an equal volume of phosphate buffer; A3: replace the test solution and α-amylase solution with equal volumes of distilled water and phosphate buffer, respectively.

[0074] 2. Inhibition of α-glucosidase activity in vitro by non-starch polysaccharides from white lotus (1) Solution preparation: 1 U / mL α-glucosidase solution was prepared with 0.1 mol / L phosphate buffer at pH 6.8 and stored at -20°C; 0.3765 g of white lotus non-starch polysaccharide sample was accurately weighed and added to an appropriate amount of phosphate buffer, stirred thoroughly until completely dissolved, and then diluted to 50 mL with phosphate buffer. After shaking, a 25 mmol / L PNPG solution was obtained; 0.53 g of Na2CO3 powder was weighed and added to an appropriate amount of distilled water, stirred thoroughly until completely dissolved, and then diluted to 50 mL to obtain a 0.1 mol / L Na2CO3 reaction termination solution. It was stored at 4°C for later use; an appropriate amount of white lotus non-starch polysaccharide sample was weighed and completely dissolved with distilled water. After dilution, a series of LSPS solutions with concentrations of 0.1, 0.25, 0.5, 1, 2, 4, and 8 mg / mL were prepared for later use. Acarbose was weighed, dissolved and diluted with distilled water to prepare a series of positive control solutions with concentrations of 0.1, 0.25, 0.5, 1, 2, 4, and 8 mg / mL for later use.

[0075] (2) Detection: The inhibition rate of LSPS on α-glucosidase activity was determined by the PNPG method, with acarbose as a positive control. 40 μL of LSPS solution and 40 μL of α-glucosidase solution were placed in a 96-well plate and reacted at 37°C for 15 min. 40 μL of PNPG solution was then added to initiate the reaction. After reacting at 37°C for 25 min, 100 μL of 0.2 mol / L sodium carbonate was added to terminate the reaction. The absorbance value (A) was measured at a wavelength of 405 nm using an enzyme reader. i ; Acarbose solution was used instead of LSPS solution, and the absorbance of acarbose was determined according to the above method. The inhibition rate was calculated as shown in formula (2), and the results are shown in Figure 5 : Inhibition rate (%) = [1-(A1-A2) / (A0-A3)] × 100 (2) Wherein, A0: replace the test solution with an equal volume of distilled water; A2: replace the α-glucosidase solution with an equal volume of phosphate buffer; A3: replace the test solution and α-glucosidase solution with equal volumes of distilled water and phosphate buffer, respectively.

[0076] Depend on Figure 4 and Figure 5 It can be seen that non-starch polysaccharides from white lotus have a certain inhibitory effect on α-amylase and α-glucosidase, and have a better inhibitory effect at high concentrations. At a concentration of 8 mg / mL, the inhibition rates of LSPS on α-amylase and α-glucosidase were 62.56±2.01% and 87.06±0.24%, respectively, indicating that non-starch polysaccharides from white lotus may have a certain potential to lower blood sugar.

[0077] 3. Analysis of the effect of non-starch polysaccharides from white lotus on improving comprehensive metabolic indicators in T2DM model mice 3.1 Establishment of T2DM model (1) Acclimation period: 36 C57BL / 6 male mice weighing 22-26 g were selected and housed individually under controlled conditions of 25±2°C, 50±5% relative humidity, and a 12 / 12 h day / night cycle. During this period, the mice had free access to water and food.

[0078] (2) High-fat and high-sugar diet modeling period: After a one-week adaptation period, the mice were randomly divided into a standard diet group (12 mice, maintenance feed) and a high-fat and high-sugar diet group (36 mice, D12492 feed). This stage lasted for 6 weeks.

[0079] (3) Type 2 diabetes mellitus (T2DM) model establishment period: After the model establishment period, mice in the high-fat and high-sugar diet group were fasted for 12 hours and then intraperitoneally injected with low-temperature streptozotocin (STZ, dose of 75 mg / kg body weight) for 5 consecutive days. One week after STZ injection, the fasting blood glucose index (FBG) was measured. If FBG was ≥11.1 mmol / L and maintained stably for more than 2 weeks, the model was successfully established. Mice in the T2DM model group were fed D12492 feed throughout the experimental period. Mice on the standard diet were given an equal amount of 0.5 mol / L citric acid buffer.

[0080] 3.2 Experimental Grouping and Treatment Diabetic mice were randomly divided into three groups, 12 in each: a diabetes model group (DC), a metformin positive control group (Met, Met dosed at 200 mg / kg body weight), and a white lotus non-starch polysaccharide group (LSPS, LSPS dosed at 200 mg / kg body weight). Mice fed a standard diet served as a blank control group (NC). During the feeding period, all mice had free access to food and water. Each group received oral gavage once daily at 9:00 AM for 5 consecutive weeks. The dosage of oral gavage for each group is shown in Table 1. After the feeding period, all mice were fasted for 12 hours and then sacrificed.

[0081] Table 3 Animal gavage treatment method

[0082] 3.3 Index determination 3.3.1 Weight During the experiment, the body weight of mice in each group was recorded once a week.

[0083] Weight loss is one of the typical symptoms of T2DM mice. After successful modeling, the weight changes of mice during the experiment are as follows: Figure 6 As shown, the body weight of mice in the diabetic model group decreased compared to normal mice. The body weight of mice in the NC group steadily increased throughout the experiment, while the DC group showed the opposite trend, exhibiting significant weight loss. This may be because STZ releases toxic nitrogen oxides, which cause pancreatic β-cell necrosis. Damage to pancreatic β-cells leads to endocrine disorders and dyslipidemia, further creating a negative nitrogen balance and causing weight loss. After 5 weeks of oral administration, the body weight of mice in both the LSPS and Met groups showed a trend of initial decrease followed by an increase, indicating that LSPS intervention can, to a certain extent, prevent the continued weight loss of T2DM mice.

[0084] 3.3.2 Fasting blood glucose During the experiment, mice in each group were fasted for 12 h each week, and then their tails were cut to collect blood. The changes in fasting blood glucose levels in each group were recorded using a blood glucose meter.

[0085] Fasting blood glucose level is the main criterion for diagnosing diabetes and evaluating the effect of drugs on lowering blood glucose. Figure 7 As can be seen, the blood glucose levels of mice in the NC group remained essentially unchanged and were significantly lower than those of the other three groups of diabetic mice (P<0.05). After 5 weeks of LSPS treatment, the FBG level in the DC group increased by 19.91% compared to the blood glucose level at the time of successful modeling. This may be attributed to the persistent effect of STZ, which can continuously damage pancreatic β-cell function. After intervention, the blood glucose levels of T2DM mice in the LSPS and Met groups decreased significantly, indicating that LSPS has a significant effect on blood glucose regulation.

[0086] 3.3.3 Oral glucose tolerance test One week before the end of feeding, an oral glucose tolerance test (OGTT) was performed. Mice in each group were fasted overnight but not water deprived. A 2 g / kg glucose solution was then gavaged orally. Blood was collected by tail clipping at 0, 15, 30, 60, 90, and 120 minutes, and real-time blood glucose levels were measured. A blood glucose graph was constructed with blood glucose levels plotted against time, and the area under the blood glucose curve (AUC) was calculated for each experimental group.

[0087] The oral glucose tolerance of mice was evaluated based on the area under the blood glucose curve (AUC). Figure 8 OGTT is an indicator for evaluating insulin secretion function. Figure 8 As shown in (A), glucose ingestion caused a rapid increase in blood glucose levels in all mice. In normal mice, blood glucose levels peaked at 15 minutes, then gradually declined, reaching near recovery by 60 minutes. Because pancreatic β-cell damage in T2DM mice leads to impaired blood glucose regulation, the blood glucose levels of T2DM mice in the untreated DC group were significantly higher than those in the NC group. Peak blood glucose levels in the LSPS group were slightly lower than those in the DC group, but the difference was not significant. At 120 minutes, blood glucose levels in the LSPS group remained higher than those in the NC group, but showed a downward trend compared to the DC group, suggesting that LSPS may have a slight effect on blood glucose regulation.

[0088] Impaired glucose tolerance is the intermediate state between normal glucose tolerance and type 2 diabetes. It is also one of the main criteria for diagnosing prediabetes. Figure 8 (B) AUC results: The DC group had significantly higher AUC values than the NC group. Compared with the DC group, metformin and LSPS significantly reduced the AUC in T2DM mice. This result suggests that LSPS significantly accelerates glucose metabolism in the blood and improves glucose tolerance in diabetic mice.

[0089] 3.3.4 Fasting insulin tolerance test Three days before the end of feeding, an insulin tolerance test (ITT) was performed. Mice were fasted for 6 hours and then injected with 1.0 U / kg body weight of insulin. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes after insulin injection, and the area under the blood glucose curve (AUC) was calculated to assess insulin tolerance. The results are shown in Figure 2. Figure 9 .

[0090] Insulin tolerance test (ITT) is a method to assess insulin sensitivity in mice. By measuring the blood glucose levels of mice at different time points after insulin injection, we can understand the regulatory effect of insulin on blood glucose and thus determine whether the mice have insulin resistance. Figure 9 As shown in (A), blood glucose levels in normal mice dropped rapidly after insulin ingestion, reaching a minimum at 30 minutes before gradually rising and returning to their original level by 120 minutes. However, in the DC group, T2DM mice exhibited insulin resistance, and their blood glucose levels barely decreased after insulin injection. This is because insulin resistance reduces cellular sensitivity to insulin, preventing insulin from effectively taking effect. Compared to the DC group, T2DM mice in the Met and LSPS groups showed similar blood glucose level changes after insulin injection as those in the NC group.

[0091] Depend on Figure 9 (B) The AUC results show that the AUC value of the DC group was significantly higher than that of the NC group. Compared with the DC group, metformin and LSPS significantly reduced the AUC of T2DM mice. The smaller the AUC, the higher the insulin sensitivity of the mice and the better the insulin regulation of blood sugar. This result shows that LSPS significantly improved the insulin sensitivity of T2DM mice.

[0092] 3.3.5 Serum insulin level measurement and insulin resistance index evaluation After gavage, mice were fasted for 8 h, their eyeballs were removed and blood was collected. The blood was centrifuged at 3500 r / min for 15 min, and serum was collected. The mouse insulin enzyme-linked immunosorbent assay (ELISA) kit was used to measure the mouse serum insulin level (FINS). The insulin resistance index (HOMA-IR) was calculated according to the following formula (3). The results are shown in Figure 10 .

[0093] HOMA-IR = [serum insulin concentration (μIU / mL) × fasting blood glucose concentration (mmol / L)] / 22.5 (3) Insulin is the only hormone in the human body that regulates blood sugar concentration. The main cause of type 2 diabetes is relatively insufficient insulin secretion or insulin resistance. Promoting the body's insulin secretion and enhancing insulin sensitivity are important strategies for treating type 2 diabetes. Figure 10(A) Compared with the NC group, the serum insulin content of mice in the DC group increased by 258.55%, indicating that type 2 diabetic mice may have obvious insulin resistance. The intervention of metformin and LSPS significantly reduced the serum insulin content.

[0094] HOMA-IR is another reliable indicator for evaluating insulin resistance. A large number of studies have shown that insulin resistance is associated with a variety of diseases, including obesity, metabolic fatty liver, cardiovascular disease, cancer, etc. Figure 10 (B) As shown, the HOMA-IR value in the DC group was significantly increased by nearly 2.6 times compared with the NC group. Compared with the DC group, the HOMA-IR values in the LSPS and Met groups were significantly decreased. These results indicate that LSPS can improve the insulin sensitivity of T2DM mice, thereby reducing their insulin resistance.

[0095] 3.3.6 Determination of mouse glycosylated protein (GSP) levels After gavage, mice were fasted for 8 h, their eyeballs were removed and blood was collected. The blood was centrifuged at 3500 r / min for 15 min, and serum was collected. The mouse serum GSP level was measured using a mouse Glycated Serum Protein (GSP) ELISA kit. The results are shown in Figure 11 .

[0096] GSP is formed by non-enzymatic glycosylation reaction between glucose in the blood and the N-terminus of albumin and other protein molecules. Since the half-life of albumin in serum is about 21 days, glycated serum protein reflects the average blood sugar level in the past 2-3 weeks and is currently recognized as a good indicator for evaluating diabetic drugs. Figure 11 The GSP value in the DC group was significantly increased by nearly 100% compared with the NC group. Compared with the DC group, after 5 weeks of oral administration of white lotus non-starch polysaccharide or metformin, the GSP content in the blood of T2DM mice in the LSPS and Met groups was significantly reduced, with significant differences between the groups. This indicates that white lotus non-starch polysaccharide has a good effect on controlling blood sugar in diabetic mice.

[0097] 3.3.7 Determination of mouse intestinal flora in the in vivo hypoglycemic experiment After gavage, the feces of mice in each group were collected to determine the fecal microbial composition. The total DNA in the fecal samples of mice in each group was extracted using a fecal genomic DNA extraction kit. After quality inspection and quantitative analysis, the 16S rRNA V3-V4 region was PCR amplified. After the PCR reaction was completed, the amplified products were purified and recovered using an agarose gel DNA recovery kit. The DNA samples were quantified using a Qubit 3.0 fluorometer, and the libraries were mixed and prepared. The library quality was evaluated using an Agilent 2100 bioanalyzer, and finally sequenced using the Illumina MiSeq platform. Based on the sequencing quality, the paired-end reads were quality controlled and filtered, and spliced based on their overlapping parts to generate optimized data after quality control. Then, noise reduction technologies such as DADA2 / Deblur were used to process these data to obtain ASV (amplicon sequence variant) representative sequences and their abundance information. Based on the comparison of ASV representative sequences and abundance information, species taxonomy analysis was performed on the Silva database, and the diversity and species differences of the intestinal flora were calculated. The results are shown in Figure 12-13 .

[0098] The figure shows the changes in the intestinal flora of mice in different treatment groups. Among multiple bacterial groups, the abundance of the DC group was significantly lower than that of the blank control group (NC), indicating that diabetes may have a negative impact on the diversity and abundance of the intestinal flora of mice. Compared with the NC group, the intestinal flora of mice in the DC group changed significantly at the phylum level, with Bacteroidetes ( Bacteroidetes ), Proteobacteria ( Proteobacteria ), Verrucomicrobia ( Verrucomicrobia ) and Deferribacteria ( Deferribacteres ) increased in relative abundance, while Firmicutes ( Firmicutes ) and Actinobacteria ( Actinobacteria ) decreased in relative abundance. These changes may be related to metabolic disorders and intestinal environmental changes caused by diabetes, which may further aggravate disease progression and affect metabolism, inflammation and barrier function. In the Met group, the relative abundance of Bacteroidetes and Deferrococcus decreased significantly, while the relative abundance of Verrucomicrobia increased significantly, indicating that metformin alleviates diabetic metabolic disorders by reshaping the intestinal microecology. Compared with the DC group, the relative abundance of Firmicutes and Actinobacteria increased significantly in the LSPS group, while the relative abundance of Bacteroidetes, Proteobacteria, Verrucomicrobia and Deferrococcus decreased significantly, indicating that polysaccharides promote the proliferation of Firmicutes through prebiotic effects, thereby enhancing intestinal barrier function. In addition, polysaccharides promote the proliferation of Firmicutes through a prebiotic effect that is independent of Akkermansia The results showed that the non-starch polysaccharide of white lotus can improve the health of diabetic mice by regulating intestinal flora.

[0099] 3.3.8 Determination of Short-Chain Fatty Acid Content in Mouse Feces Weigh the contents of the cecum and record the weight, then add 1.0 mL of normal saline and grind at high speed. After mixing, centrifuge at 13,000 rpm / min for 5 min at 4°C, filter the supernatant through a 0.22 μm water filter, aspirate about 0.5 mL of the filtrate, and record the exact volume. After acidification with 0.2 mL of 10% (v / v) sulfuric acid solution, add 0.4 mL of anhydrous ether to extract SCFA, centrifuge at 13,000 rpm / min for 2 min, collect the supernatant, pass through a 0.22 μm organic filter, and place in a sample bottle. Detection was performed using an Agilent 7890B gas chromatograph. Combined with the external standard method, the content of short-chain fatty acids in each sample was calculated. The results are shown in the table. Figure 14 .

[0100] Changes in SCFAs content are closely related to the occurrence and development of T2DM. Studies have found that the number of SCFAs-producing bacteria in the intestines of T2DM patients and model mice is reduced, resulting in a decrease in SCFAs levels. At the same time, short-chain fatty acids are the main metabolites of dietary fiber fermentation by intestinal flora, and their content can reflect the metabolic activity and composition changes of intestinal flora. Among them, butyrate can stimulate intestinal L cells to release glucagon-like peptide-1 (GLP-1), thereby enhancing insulin secretion and inhibiting glucagon release, effectively regulating blood sugar to a lower level. Propionic acid reduces endogenous glucose production by inhibiting glucose-6-phosphatase activity in the liver. Isobutyric acid may be involved in regulating the energy metabolism, lipid metabolism and other processes of diabetic mice, and may regulate fat metabolism by affecting the differentiation and function of fat cells. Isovaleric acid may interfere with the insulin signaling pathway by affecting the metabolic signal transduction between the intestine and the body, affecting the uptake and utilization of glucose by cells, etc. From Figure 14 Changes in the content of different SCFAs can be observed in different groups. The levels of acetic acid, propionic acid, and valeric acid in the cecal contents of mice in the DC group were significantly lower than those in the control group, indicating that diabetes may cause an imbalance in the intestinal flora and reduce the production of SCFAs; after metformin intervention, the valeric acid content of the Met group increased significantly; after intervention with white lotus non-starch polysaccharides, the six SCFAs such as acetic acid, propionic acid, and isobutyric acid in the LSPS group were significantly higher than those in the DC group. This shows that white lotus non-starch polysaccharides have a certain positive effect on improving the intestinal flora of diabetic mice and increasing the production of SCFAs. White lotus non-starch polysaccharides may improve the health of diabetic mice by regulating the intestinal flora and increasing the content of SCFAs.

[0101] In summary, the present invention extracts a new white lotus non-starch polysaccharide from white lotus. This polysaccharide can regulate metabolic abnormalities in multiple dimensions, exert a hypoglycemic effect, and has broad application prospects in the prevention and treatment of diabetes.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A white lotus non-starch polysaccharide, characterized in that The white lotus non-starch polysaccharide is a pyranose polysaccharide with an α-configuration, composed of (0.57%±0.47%) fucose, (11.75%±0.53%) rhamnose, (1.37%±0.73%) arabinose, (20.18%±0.19%) galactose, (49.44%±0.28%) glucose, (3.43%±0.35%) xylose, (2.93%±0.14%) mannose, (10.19%±0.12%) galacturonic acid and (0.13%±0.1%) glucuronic acid, with a molecular weight of Mw 145KDa.

2. The method for preparing the white lotus non-starch polysaccharide according to claim 1, characterized in that: The preparation method comprises the following steps: Step 1: Wash and core the white lotus, dry and grind it to obtain coarse white lotus powder; Step 2: adding water to the crude white lotus powder obtained in step 1 and mixing to obtain a suspension; subjecting the suspension to pressurized water-eddy current pulse electric field combined extraction, solid-liquid separation, and collecting the supernatant to obtain a white lotus extract; Step 3: adding immobilized enzyme microspheres to the white lotus extract obtained in step 2 at a ratio of 40-60 g immobilized enzyme microspheres per 100 g crude white lotus powder, performing enzymatic hydrolysis at 50-60° C. until the iodine solution color reaction of the white lotus extract turns colorless or light yellow, thereby obtaining an enzymatic hydrolyzate; performing solid-liquid separation on the enzymatic hydrolyzate, taking the supernatant for alcohol precipitation, and collecting the precipitate; removing residual ethanol from the precipitate, and drying to obtain crude white lotus polysaccharide; the immobilized enzyme microspheres are enzyme-biomass-based gel complexes, and the enzymes are one or more enzymes capable of degrading white lotus starch or cellulose; Step 4: dissolving the crude polysaccharide of white lotus in water to prepare a crude polysaccharide solution; subjecting the crude polysaccharide solution to column chromatography, wherein the column chromatography uses a D101 macroporous resin column as a stationary phase and distilled water as an elution solvent; when the molish reaction of the eluate is negative, the elution is stopped, and the collected eluate is concentrated and dried to obtain the crude non-starch polysaccharide of white lotus; Step 5: dissolving the crude non-starch polysaccharide of white lotus in water to prepare a non-starch polysaccharide solution, filtering the obtained filtrate and performing gel filtration chromatography, wherein the gel filtration chromatography adopts a Sephacryl S-400 HR gel filtration column, the elution solvent is 0.1M sodium chloride solution, and the eluate corresponding to the polysaccharide peak in the absorbance curve at 490 nm is collected; the eluate is dialyzed through an 8000-12000 Da dialysis bag, and the dialyzed retentate is dried to obtain the non-starch polysaccharide of white lotus.

3. The preparation method according to claim 2, characterized in that The crude white lotus powder and water in step 2 are mixed at a material-liquid ratio of 1:15-20 (g / mL), and soaked until the soluble components are fully dissolved to obtain a suspension; the suspension is treated at 110°C, 0.4 MPa, and 200 rpm for 30-40 minutes; and then a pulsed electric field of 15 kV / cm and an eddy current of 30 kHz are applied for 10-20 minutes.

4. The preparation method according to claim 2, characterized in that The preparation method of the immobilized enzyme microspheres described in step 3 includes: dissolving the enzyme in phosphate buffer to prepare an enzyme solution; adding biomass-based gel microspheres with a diameter of 2-3 mm to the enzyme solution, shaking and adsorbing them thoroughly, and then adding a cross-linking agent to form a covalently bond-stabilized enzyme-biomass-based gel complex; after cross-linking, centrifuging and collecting the immobilized enzyme microspheres, removing unbound free enzyme and residual cross-linking agent, and obtaining immobilized enzyme microspheres.

5. The preparation method according to claim 4, characterized in that The enzyme is a combination of amylase and cellulase; the mass ratio of amylase to cellulase in the enzyme solution is 3:1, and the concentration of the enzyme in the enzyme solution is 10-20 mg / mL; biomass-based gel microspheres are added to the enzyme solution at a ratio of 2 mL enzyme solution / g; and / or The biomass-based gel microspheres are sodium alginate-based gel microspheres; and the cross-linking agent is genipin.

6. The preparation method according to claim 4, characterized in that The oscillating adsorption is performed at 150 rpm for 8 hours; and / or The cross-linking conditions are 37° C., protected from light, 50 rpm, and time for 24 h.

7. The preparation method according to claim 2, characterized in that In step 3, the immobilized enzyme microspheres are added to the white lotus extract at a ratio of 60 g immobilized enzyme microspheres per 100 g crude white lotus powder; and / or The alcohol precipitation comprises the following steps: adding ethanol to the supernatant to make the mass percentage of ethanol in the solution reach 70%-80%, stirring sufficiently, and then standing until the weight of the precipitate no longer increases.

8. The preparation method according to claim 2, characterized in that The concentration of the crude polysaccharide solution in step 4 is 10-20 mg / mL, and elution is performed more than 8 hours after adsorption; and / or The concentration of the non-starch polysaccharide solution in step 5 is 20 mg / mL, the flow rate of the elution solvent is 0.8 mL / min, and the eluate with an elution time between 11.8 and 15.3 min is collected.

9. Use of the white lotus non-starch polysaccharide according to claim 1 and the white lotus non-starch polysaccharide prepared according to the preparation method according to claims 2 to 8 in food, health food and medicine.

10. The use according to claim 9, characterized in that include Application in health foods that help maintain blood sugar health or help regulate intestinal flora; Application in medicines for lowering blood sugar, alleviating insulin resistance or reducing glycosylated serum protein levels; Application in medicines for regulating intestinal flora.

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