Preparation method and application of miscanthus araboxylan

Abstract

The invention provides a preparation method and application of miscanthus arabinoxylan, and particularly relates to application of miscanthus arabinoxylan in preparation of antioxidant, hypoglycemic or lipid-lowering products and application of miscanthus arabinoxylan in preparation of intestinal flora homeostasis regulating products. The Chinese silvergrass-sourced arabinoxylan has the functions of reducing blood sugar, adsorbing bile acid and resisting oxidation in vitro, and can protect the growth of bifidobacterium longum by adsorbing bile acid, thereby playing a probiotic role.

Description

Technical Field

[0001] This invention relates to the field of natural products, specifically to a method for preparing and applying Miscanthus arabinoxylan. Background Technology

[0002] Currently, diseases such as overweight, obesity, diabetes, and hyperlipidemia are mainly treated through dieting, increased exercise, medication, and surgery. However, these treatments often have side effects, such as long treatment cycles and high costs. Therefore, how to prevent or intervene in the occurrence of these chronic diseases has become an urgent social problem to be solved.

[0003] Dietary fiber is a type of carbohydrate that is not directly digested and absorbed by the small intestine but can be fermented in the large intestine, providing health benefits. Arabicoxylan is a major non-cellulose dietary fiber found in grains and grasses. As a common dietary fiber found as a byproduct of grains, arabinoxylan has a wide range of sources (such as corn, rye, barley, oats, sorghum, wheat, and rice). However, arabinoxylan extracted from different raw materials or using different methods has its own unique structural characteristics. These arabinoxylans with different structures also exhibit different functional characteristics.

[0004] Bile acids are natural emulsifiers involved in the digestion and absorption of lipids and fat-soluble vitamins. In healthy individuals, the bile acid pool maintains stability through enterohepatic circulation. However, some diseases can lead to dysregulation of enteroendocrine and neurohormonal signaling pathways, resulting in abnormal bile acid pools. Gut microbiota plays a crucial role in bile acid metabolism, but excessive bile acids can disrupt gut microbiota stability, causing colitis and even colon cancer. In the adult gut, *Bifidobacterium longum* is a representative probiotic. Due to the amphiphilic structure of bile acids, they can interact with membrane phospholipids, thereby disrupting bacterial cell membranes and exerting bactericidal activity. This mechanism allows bile acids to inhibit the growth and proliferation of various bacteria, including *Bifidobacterium longum*.

[0005] Miscanthus, as an energy crop, is often explored for its biomass value, such as the production of ethanol through fermentation. Miscanthus is rich in bioactive substances such as cellulose, hemicellulose, vitamins, proteins, and polysaccharides, possessing high economic and nutritional value. Numerous studies have reported the physicochemical functions and health-promoting effects of hemicellulose polysaccharides; however, there are currently no definitive research reports on the regulatory effects of Miscanthus arabinoxylan on blood sugar, lipid-lowering effects, antioxidant activity, or its effects on microorganisms. Summary of the Invention

[0006] The present invention aims to at least partially solve one of the technical problems in the above-mentioned technology, namely, to provide a method for preparing and applying Miscanthus arabic xylan, which has the functions of lowering blood sugar, adsorbing bile acids, and anti-oxidation in vitro. It can also protect the growth of Bifidobacterium longum by adsorbing bile acids, while being specifically degraded itself, thereby exerting a beneficial effect.

[0007] Therefore, in a first aspect of the invention, the invention proposes the application of arabinoxylan from Miscanthus sinensis in the preparation of products with antioxidant, hypoglycemic, or lipid-lowering properties.

[0008] According to the application of the present invention, the arabinoxylan from Miscanthus sinensis exhibits hypoglycemic, hypolipidemic, and antioxidant activities in vitro. It can be used as a potential prebiotic to regulate blood sugar and blood lipids, as well as exert antioxidant effects, making it applicable to the development of dietary supplements to support the body's metabolic health.

[0009] In a second aspect of the invention, the invention proposes the application of arabinoxylan from Miscanthus sinensis in the preparation of products that regulate intestinal flora homeostasis.

[0010] According to the application of the present invention, the arabinoxylan from Miscanthus sinensis can protect the growth viability of long-chain Bifidobacteria in a bile acid environment, maintain the content of beneficial metabolites, and has the potential for combined use with probiotics.

[0011] Optionally, the product is used to protect the growth viability of Bifidobacterium longum in a bile acid environment.

[0012] Optionally, the product is used to maintain the levels of beneficial metabolites produced by long-chain bifidobacteria.

[0013] Furthermore, the beneficial metabolites include short-chain fatty acids and lactic acid.

[0014] In a third aspect of the invention, the invention provides a method for preparing the above-mentioned Miscanthus arabic xylan, wherein the arabic xylan is prepared by using Miscanthus as raw material, through alkali treatment, ultrasonic-assisted extraction, water bath heating, alcohol precipitation, and vacuum freeze-drying.

[0015] Furthermore, the Miscanthus arabic xylan is prepared by a method including the following steps:

[0016] The raw material of Miscanthus sinensis is crushed and sieved to obtain Miscanthus sinensis powder;

[0017] The powdered Miscanthus sinensis was treated with alkali by adding NaOH with a mass-volume concentration of 12% at a material-to-liquid ratio of 1:20.

[0018] The alkali-treated sample solution was extracted using an ultrasonic-assisted water bath to obtain the extract.

[0019] Centrifuge the extract and collect the supernatant;

[0020] Add ethanol to the supernatant for alcohol precipitation, and adjust the final ethanol concentration to 70%.

[0021] The ethanol-precipitated solution was centrifuged and the precipitate was collected and freeze-dried under vacuum to obtain the soluble dietary fiber from Miscanthus sinensis.

[0022] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0023] Figure 1 The above is an HPLC chromatogram of the monosaccharide composition of Miscanthus arabic xylan according to an embodiment of the present invention.

[0024] Figure 2 To illustrate the antioxidant effect of Miscanthus arabic xylan according to embodiments of the present invention;

[0025] Figure 3 The hypoglycemic effect of Miscanthus arabinoxylan according to an embodiment of the present invention;

[0026] Figure 4 To illustrate the lipid-lowering effect of Miscanthus arabic xylan according to embodiments of the present invention;

[0027] Figure 5 The growth curves and viable counts of long-chain Bifidobacteria were obtained by fermenting Miscanthus arabic xylan in environments containing and without bile acids, according to embodiments of the present invention.

[0028] Figure 6 The content of acetic acid produced by fermenting long-chain Bifidobacterium in environments containing and without bile acids, according to embodiments of the present invention;

[0029] Figure 7 The content of lactic acid produced by fermentation of *Miscanthus arabiculatus* in environments containing and without bile acids, according to embodiments of the present invention;

[0030] Figure 8 The pH changes and total sugar degradation rate of *Miscanthus arabinoxylan* fermented with *Bifidobacterium longum* in environments containing and without bile acids, according to embodiments of the present invention;

[0031] Figure 9 The morphological changes of *Miscanthus arabicxylan* were determined by fermenting it with *Bifidobacterium longum* in environments containing and without bile acids for 48 hours, according to an embodiment of the present invention. Detailed Implementation

[0032] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0033] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0034] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0035] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0036] Example 1: Preparation of arabinoxylan from Miscanthus sinensis

[0037] (1) Crushing: Crush the freshly picked Miscanthus sinensis with a crusher and pass it through a 40-mesh sieve to obtain Miscanthus sinensis powder.

[0038] (2) Alkali treatment: Add NaOH with a mass-volume concentration of 12% to the Miscanthus powder at a material-to-liquid ratio of 1:20 (v:v) and mix well to obtain the sample solution.

[0039] (3) Ultrasonic water bath treatment: The sample solution from step (2) was ultrasonicated in a water bath for 15 minutes, and then placed in a 70℃ water bath for 3.5 hours to obtain the extract.

[0040] (4) Centrifugation: Centrifuge the extract from step (3) at room temperature and 4000g for 20 minutes. Collect the supernatant after centrifugation.

[0041] (5) Adjust pH: Adjust the pH of the supernatant to 7 with HCl.

[0042] (6) Ethanol precipitation: Add anhydrous ethanol to the supernatant of step (5) until the sample solution contains 70% ethanol, stir for 30 min and then place at 4℃ overnight for alcohol precipitation.

[0043] (7) Centrifugation: Centrifuge the solution after alcohol precipitation and collect the precipitate;

[0044] (8) Freeze-drying: The precipitate obtained in step (7) was freeze-dried under vacuum (cold hydrazine temperature -70℃, 0MPa, 48h) to obtain arabinoxylan.

[0045] Example 2: Determination of monosaccharide composition of arabinoxylan from Miscanthus sinensis

[0046] (1) Hydrolysis of sample: Weigh 5 mg (±0.05 mg) of the Miscanthus arabinoxylan sample obtained in Example 1, add 1 mL of 2 M TFA acid solution, and heat at 121 °C for 2 hours. Purge with nitrogen and dry. Add 3 mL of methanol to wash and dry again, repeating the methanol washing 2-3 times. Add 5 mL of sterile water to dissolve.

[0047] (2) Pre-column derivatization: 5 mg each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and glucosamine galactose hydrochloride monosaccharides, and 10 mg of fucose were dissolved and diluted to 10 mL in a volumetric flask to prepare a standard stock solution. This stock solution was then serially diluted, filtered through a 0.22 μm microporous membrane, and placed into a sample vial. 0.2 mL of the monosaccharide standard solution or hydrolysate of arabinoxylan was added to a stoppered conical centrifuge tube. 0.2 mL of 0.5 mol / L sodium hydroxide solution and 0.5 mL of 0.5 mol / L PMP methanol solution were added. After vortexing, the mixture was reacted in a 70℃ water bath for 1 h. After the reaction was complete, 0.2 mL of 0.5 mol / L hydrochloric acid was added to neutralize the added sodium hydroxide. 1 mL of chloroform was added, and the mixture was vortexed three times to remove excess PMP. After discarding the chloroform layer, 0.3 mL of the chloroform layer was added and diluted to 1 mL with water.

[0048] (3) Chromatographic conditions: Thermo U3000 liquid chromatography system, ZORBAX EclipseXDB-C18 column, mobile phase is acetonitrile: phosphate buffer (potassium dihydrogen phosphate 12 g / L, pH adjusted to 6.8 by 2M NaOH) isocratic elution, volume ratio of acetonitrile to phosphate buffer is 17:83, flow rate is 0.8 mL / min, column temperature is 30℃, detection wavelength is 250 nm, injection volume is 10 μL, the determination is performed using mixed monosaccharide standard and derivatized products of Miscanthus arabinoxylan hydrolysis sample, and well separated chromatographic peaks are obtained.

[0049] (4) Analysis of the results regarding monosaccharide composition: Figure 1 It can be seen that the arabinoxylan from Miscanthus sinensis is represented by MIS. Figure 1 'a' is the chromatogram of the standard quantified using the external standard method, corresponding to the peak time and peak area. Figure 1The monosaccharide composition of arabinoxylan from *Miscanthus sinensis* was determined to be 0.29% Man, 0.35% GlcN, 0.78% Rha, 0.57% GlcA, 0.26% GalA, 10.17% Glc, 2.18% Gal, 70.09% Xyl, and 15.32% Ara. Therefore, the main component of *Miscanthus sinensis* arabinoxylan is arabinoxylan.

[0050] Example 3: Antioxidant function of Miscanthus arabinoxylan

[0051] (1) Preparation of VC solution: Weigh 0.1g of ascorbic acid (VC) reagent, dissolve it in distilled water, quantitatively transfer it to a 10mL volumetric flask and make up to volume to obtain a VC solution with a concentration of 10mg / mL.

[0052] (2) Preparation of sample solution: Weigh 0.5g of the arabinoxylan obtained in Example 1 and dilute it with pure water in a 50mL volumetric flask to prepare a 10mg / mL sample solution for the experiment.

[0053] (3) Hydroxyl radical scavenging ability

[0054] Accurately weigh 0.0348 g of ferrous sulfate heptahydrate powder and dilute it to 50 mL in a volumetric flask with ultrapure water for later use.

[0055] Accurately weigh 0.06906 mg of salicylic acid powder and dilute it to 50 mL in a volumetric flask with ultrapure water for later use.

[0056] Accurately pipette 10 μL of hydrochloric acid into a 10 mL centrifuge tube, add 4.79 mL of water, and shake well before use.

[0057] Accurately pipette 10 μL of 30% hydrogen peroxide into a 10 mL centrifuge tube, add 9.78 mL of water, and shake well before use.

[0058] 50 μL of salicylic acid (10 mmol / L), 50 μL of hydrochloric acid (2.5 mmol / L), 50 μL of ferrous sulfate (2.5 mmol / L), 50 μL of H₂O₂ (10 mmol / L), and 50 μL of 10 mg / mL Miscanthus arabic xylan sample were sequentially added to a 96-well plate and vortexed. After reacting at 37℃ for 30 min, the absorbance (As) at 510 nm was measured using a microplate reader. Ultrapure water was used as a blank control (A0) instead of the sample, and ultrapure water was used as a sample control instead of H₂O₂ (AS0). 10 mg / mL Vitamin C was used as a positive control instead of the sample. The formula for calculating the hydroxyl radical scavenging rate of the sample is as follows:

[0059]

[0060] In the formula: AS is the absorbance of the sample group; AS0 is the absorbance of the sample control group; A0 is the absorbance of the blank group.

[0061] (2) Superoxide anion free radical scavenging ability

[0062] 100 μL of a 10 mg / mL Miscanthus arabinoxylan sample solution was added to 300 μL of 50 mmol / L Tris-HCl buffer (pH 8.2) and incubated at 30 °C for 20 min. Then, 300 μL of 7 mmol / L pyrogallol was added and the reaction was allowed to proceed for 10 min. Finally, 100 μL of concentrated HCl was added to terminate the reaction. The mixture was centrifuged at 4000 rpm for 20 min, and the supernatant was collected. The absorbance was measured at 420 nm, with 10 mg / mL Vitamin C used as a positive control. The formula for calculating the superoxide anion radical scavenging rate is as follows:

[0063]

[0064] In the formula: AS is the absorbance of the sample group; AS0 is the absorbance of the sample control group; A0 is the absorbance of the blank group.

[0065] (3) DPPH free radical scavenging ability

[0066] Accurately weigh 6.3 mg of DPPH powder and dilute to 100 mL in a brown volumetric flask with anhydrous ethanol to obtain a DPPH solution with a concentration of 0.16 mmol / L. Store the solution protected from light for later use.

[0067] 100 μL of 10 mg / mL Miscanthus arabinoxylan sample was added to a 96-well plate, along with 100 μL of DPPH solution. The plate was shaken well and reacted in the dark for 30 minutes. The absorbance (AS) at 517 nm was then measured using a microplate reader. A blank experiment was performed using 100 μL of ultrapure water instead of the test solution. A 10 mg / mL Vitamin C sample solution was used as a positive control. The DPPH free radical scavenging rate was calculated using the following formula:

[0068]

[0069] In the formula: AS is the absorbance of the sample group; AS0 is the absorbance of the sample control group; A0 is the absorbance of the blank group.

[0070] (4) ABTS free radical scavenging ability

[0071] Accurately weigh 10 mg of ABTS powder into an Erlenmeyer flask, add 2.6 mL of ultrapure water, and sonicate for 5 min to dissolve.

[0072] Accurately weigh 2 mg of potassium persulfate (K2S2O8) into an Erlenmeyer flask, add 3 mL of ultrapure water, and sonicate for 5 min to dissolve.

[0073] Mix equal volumes of 7 mmol / L ABTS solution with 2.45 mmol / L potassium persulfate and incubate in the dark at room temperature for 10-16 hours to obtain ABTS stock solution. Dilute the ABTS stock solution 10-20 times with pure water until the absorbance at 734 nm is between 0.7 and 0.9, and store this as ABTS working solution in the dark for later use. Add 50 μL of 10 mg / mL Miscanthus arabic xylan sample solution to a 96-well plate, add 150 μL of ABTS solution, shake well, and incubate in the dark for 6 min. Measure the absorbance at 734 nm using a microplate reader. Use pure water as a blank control and 10 mg / mL VC sample solution as a positive control. The formula for calculating the ABTS free radical scavenging rate is as follows:

[0074]

[0075] In the formula: AS is the absorbance of the sample group; AS0 is the absorbance of the sample control group; A0 is the absorbance of the blank group.

[0076] (5) Analysis of the antioxidant function of arabinoxylan from Miscanthus sinensis: Vitamin C, also known as ascorbic acid, can neutralize a variety of free radicals and is widely used as a positive control in antioxidant studies due to its relatively stable chemical properties in in vitro experiments. Hydroxyl radicals (OH·) are the most reactive free radicals among active oxygen species, possessing strong electronic oxidation capabilities and capable of damaging adjacent biomolecules; superoxide anion radicals (O2·) - It plays an important role in the formation of reactive oxygen species; ABTS (2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) free radical scavenging ability is widely used as an indicator to evaluate the antioxidant capacity of plants; DPPH free radical (1,1-diphenyl-2-trinitrophenylhydrazine free radical) is a nitrogen-centered lipid-soluble free radical, which is more stable than hydroxyl radicals and superoxide radicals. Figure 2 The results showed that the hydroxyl radical scavenging capacity of an equal amount of Miscanthus arabic xylan was 42.8% that of vitamin C, the superoxide anion scavenging capacity was 15.3% that of vitamin C, the DPPH radical scavenging capacity was 69.6% that of vitamin C, and the ABTS radical scavenging capacity was comparable to that of the positive control vitamin C. This indicates that Miscanthus arabic xylan has good antioxidant function.

[0077] Example 4: Determination of the in vitro hypoglycemic effect of Miscanthus arabinoxylan

[0078] (1) Determination of α-amylase inhibition rate

[0079] Prepare a 1% (w / v) soluble starch solution using 0.05M phosphate buffer (pH 6.5). Prepare a 2 U / mL α-amylase solution using deionized water. Perform the relevant experiments according to Table 1. Incubate 50 μL of Miscanthus arabinoxylan sample solution with the starch solution at 37°C for 10 min, then add 50 μL of starch solution and continue incubation for another 10 min. For the positive control, incubate the starch solution with the same concentration of acarbose as the sample at 37°C for 10 min, then add 50 μL of starch solution and continue incubation for another 10 min. The absorbance was then measured at 540 nm using the DNS method.

[0080] Group Sample (μL) α-Amylase solution (μL) Starch solution (μL) A sample 50 50 50 Sample A (control) 50 Equal volume of deionized water 50 A control group Equal volume of deionized water 50 50 A blank control Equal volume of deionized water Equal volume of deionized water 50

[0081]

[0082] (2) Glucose binding capacity determination

[0083] Construction of glucose standard curve: Dilute glucose standard (10 mg / mL) with distilled water to 1.6, 1.0, 0.8, 0.4, 0.2, and 0.1 mg / mL, and measure absorbance at 540 nm using the DNS method.

[0084] Weigh 0.5g of dietary fiber sample and mix thoroughly with 10mL of 50mmol / L glucose solution. Incubate in a 37℃ water bath for 6h, centrifuge at 3500r / min for 20min, and collect 30μL of the supernatant. Measure the remaining glucose content in the solution using the Solarbio reagent kit's DNS method. Calculate the glucose adsorption capacity of different dietary fibers using the formula below.

[0085]

[0086] In the formula, G0 represents the glucose concentration in the solution before adsorption (mg / mL); GS represents the glucose concentration in the solution after adsorption (mmol / L); W represents the mass of dietary fiber (g); and V represents the volume of the glucose solution (mL).

[0087] Analysis of the hypoglycemic ability of Miscanthus polysaccharides: The α-amylase inhibition rate reflects the degree of inhibition of α-amylase activity by the analyte. α-Amylase, a widely distributed enzyme in organisms, primarily catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch molecules, rapidly reducing starch viscosity and breaking it down into short-chain dextrins, oligosaccharides, and small amounts of maltose and glucose. The inhibition rate of the analyte against α-amylase indicates whether the substance has the potential to lower blood sugar, providing a basis for its development as a hypoglycemic product. Acarbose, a known α-glucosidase inhibitor, was used as a positive control in the experiment. Figure 3As shown in Figure a, the α-amylase inhibition rate of Miscanthus arabicxylan was 70% of that of the positive control acarbose, indicating that Miscanthus arabicxylan has a good inhibitory effect on α-amylase and has the potential to slow down the hydrolysis and digestion of carbohydrates such as starch and disaccharides, thereby reducing blood glucose levels. Figure 3 b reflects the adsorption capacity of Miscanthus arabic xylan for glucose in glucose solution: the glucose solution concentration before the reaction was 8.95 mg / mL, and after 6 hours of adsorption reaction with 0.5 g of Miscanthus arabic xylan, the glucose concentration in the solution was only 0.47 mg / mL. This indicates that Miscanthus arabic xylan can adsorb glucose in solution, achieving a hypoglycemic effect.

[0088] Example 5: In vitro simulated gastrointestinal digestion, determination of the ability to adsorb bile acids in a simulated small intestinal environment.

[0089] (1) Preparation of in vitro digestion solution: Add 0.5 mL of 0.3 M CaCl2(H2O)2, 30 mL of 0.5 M KCl, 6 mL of 0.5 M KH2PO4, 65 mL of 1 M NaHCO3, 25 mL of 2 M NaCl, 2 mL of 0.15 M MgCl2(H2O)6, and 2 mL of 0.5 M (NH4)2CO3 to 400 mL of ultrapure water.

[0090] (2) Preparation of a mixture to simulate a human bile acid pool: In order to simulate the environment of a human bile acid pool, a total bile acid mixture containing chenodeoxycholic acid (CA), chenodeoxycholic acid (CDCA), glycocholic acid (GCA), deoxycholic acid (DCA), glycocholic acid (GCDCA) and taurocholic acid (TCA) in a mass ratio of 4:4:3:1:1:1 was used.

[0091] (3) In vitro simulated oral digestion: Weigh 0.5g of Miscanthus arabinoxylan, dissolve it fully in 2mL of ultrapure water in a 50mL test tube, and then add 2.4mL of in vitro digestion solution, 0.45mL of 75U / mL α-amylase, 0.015mL of 0.3MCaCl2(H2O)2 and 0.135mL of ultrapure water respectively. Place the test tube in a constant temperature shaking box at 37℃ and shake at 150r / min for 2min to simulate the oral digestion environment.

[0092] (4) In vitro simulated gastric digestion: After oral digestion, 4.8 mL of in vitro digestion solution, 0.4002 mL of 200 U / mL pepsin, 0.003 mL of 0.3 M CaCl2(H2O)2 and 0.5568 mL of ultrapure water were added to the test tube. The pH of the gastric environment was adjusted to 3 with 0.24 mL of 6 M HCl. The test tube was then placed in a constant temperature shaking incubator at 37 °C and shaken at 150 r / min for 2 h to simulate the gastric digestion environment.

[0093] (5) In vitro simulated small intestinal digestion: After gastric digestion, 9.6 mL of in vitro digestion solution, 3 mL of 1000 U / mL trypsin, 1.8 mL of 10 mM bile salt, 0.024 mL of 0.3 M CaCl2(H2O)2 and 1.896 mL of ultrapure water were added to the test tube. The pH of the small intestinal environment was adjusted to 7 with 0.48 mL of 6 M NaOH. The test tube was then placed in a constant temperature shaking incubator at 37 °C and shaken at 150 r / min for 2 h to simulate the small intestinal digestion environment.

[0094] (6) Dialysis: After simulating the small intestine digestion environment, all in vitro digested samples were transferred to a solution of 25 mM... Membranes were prepared using biotechnology-grade regenerated cellulose (RC) 12-14 kDa dialysis tubes. 124 mL of 50 mM phosphate buffer (pH = 7) was added to a beaker for dialysis. Dialysis was performed using a shaker at 150 rpm and 37 °C. 200 μL of dialysate was collected at 0, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h of dialysis. The permeability of total bile acids and the ratio of bile acids bound to arabinoxylan in the dialysate were determined using a Nanjing Jiancheng TBA assay kit.

[0095] (7) Analysis of the in vitro lipid-lowering ability of Miscanthus arabinoxylan: Cholesterol is converted into bile acids in the liver through a series of enzymatic reactions. These bile acids are then secreted into bile and participate in the digestion and absorption of lipids. Cholestyramine (Cho) is a polystyrene quaternary ammonium type strongly basic anion exchange resin with very strong adsorption properties. In the small intestine, it forms insoluble compounds with bile acids, thereby preventing the reabsorption of bile acids. Cholestyramine was used as a positive control in the bile acid binding capacity analysis. Figure 4 As can be seen from a, with the increase of dialysis time, the concentration of bile acids in the dialysate gradually reaches equilibrium after 12-24 hours. Figure 4 The results showed that at the endpoint of 48 hours of dialysis, cholestyramine had a binding rate of 70.80% for the added mixed bile acids, while Miscanthus arabic xylan had a binding rate of 19.22%. In contrast, the monosaccharide control group (glucose (Glc)) and the blank control group (pure water (Blank)) showed almost no binding capacity for the mixed bile acids. This adsorption capacity of Miscanthus arabic xylan helps promote cholesterol metabolism and lower serum cholesterol levels, thereby achieving a lipid-lowering effect.

[0096] Example 6: Fermentation of Bifidobacterium longum using arabinoxylan from Miscanthus sinensis

[0097] (1) Culture medium preparation: Fortified Clostridium difficile medium (RCM) served as the Glc positive control group, containing 10.0 g / L beef meal, 10.0 g / L peptone, 3.0 g / L yeast extract, 1.0 g / L soluble starch, 5.0 g / L glucose, 0.5 g / L cysteine ​​HCl, 5.0 g / L sodium chloride, and 3.0 g / L sodium acetate. The pH of the culture medium was adjusted to 6.80 ± 0.02. Before use, all culture media and vessels were autoclaved at 121°C for 15 min. RCM solid medium was RCM medium with 3% agar powder added. Carbon-free medium (Blank) was RCM medium with 5.0 g / L glucose removed, serving as the blank control group; MIS medium was RCM medium with 5.0 g / L arabinoxylan replaced by 5.0 g / L glucose. For the systems with added bile acids, an additional 10 M of mixed bile acids was added to each of the above groups. To simulate the environment of a human bile acid pool, a total bile acid mixture containing chenodeoxycholic acid (CA), chenodeoxycholic acid (CDCA), glycocholic acid (GCA), deoxycholic acid (DCA), glycochenodeoxycholic acid (GCDCA), and taurocholic acid (TCA) in a mass ratio of 4:4:3:1:1:1 was used.

[0098] (2) Activation and propagation of the strain: The strain was stored in an environment containing 25% glycerol at -80°C. Activation was initiated by placing the strain in an anaerobic incubator and maintaining it at 39°C. The stored strain was first inoculated onto RCM solid medium and cultured for about 48 hours. A single colony was taken from the solid culture and inoculated into 5 mL of RCM liquid medium and cultured for 17 hours to reach the logarithmic growth phase.

[0099] (3) Take the activated bacterial suspension in the logarithmic growth phase and inoculate it at 6% (v / v). After centrifuging the bacterial suspension at 4000×g for 10 min, collect the precipitate into RCM medium, MIS medium, carbon-free medium, and the above three groups of mediums with added mixed bile acids. Incubate the medium in an anaerobic incubator at 39℃ for 48 h.

[0100] (4) Measurement of growth: 200 μL of bacterial culture was taken from each group every two hours to detect OD. 595 Values ​​were determined. At 0, 24, and 48 hours, the bacterial strain was diluted 1 million times and inoculated onto RCM agar plates to observe its proliferation.

[0101] (5) Growth analysis: Figure 5a-5b show the growth curves for each group in the systems with and without bile acids, with the addition of bile acids indicated by +BA. *Bifidobacterium longum* grew rapidly in Glc medium, but the absorbance of the growth curve decreased significantly in Glc+BA medium. The absorbance in MIS+BA medium remained at a similar level compared to MIS medium. The absorbance in Blank and Blank+BA remained consistently low. Figure 5 c-5e reflects the viable count and viable count retention rate in the system. Viable count retention rate = ((viable count in the bile acid system / viable count in the system without bile acids) × 100%). Although *Bifidobacterium longum* can grow rapidly in media with Glc as the carbon source, the viable count retention rate in systems with added bile acids is only 15.7%-20.2%, while in media with MIS as the carbon source, the viable count retention rate is 34.6%-45.6%. In systems without any carbon source, the viable count retention rate decreases from 38.4% to 17.0%. This indicates that *Arabinopyramidan* can maintain the growth of the probiotic *Bifidobacterium longum* in systems containing bile acids.

[0102] Example 7: Determination of short-chain fatty acids during fermentation of Miscanthus arabinoxylan

[0103] (1) Sample processing: The fermentation broth at different time points in Example 6 was centrifuged (6000×g, 10min), and 160μL of supernatant was taken into a centrifuge tube. 40μL of 25% sulfuric acid was added and vortexed for 15s. After acidification and extraction, 50μL of internal standard (2-ethylbutyric acid) was added. Then, 750μL of anhydrous diethyl ether was added for extraction two to three times. The diethyl ether extracts from the three extractions were mixed and centrifuged at 10000×g and 4℃ for 5min. The upper diethyl ether extract was taken into a sample vial for testing. SCFA standard was prepared in the same way.

[0104] (2) Gas chromatography determination: SCFAs were separated using an Agilent HP-INNOWAX column (30m × 0.32mm inner diameter × 0.25μmil thickness, Agilent 19091N-113l) and a flame ionization detector. N2 was used as the carrier gas at a split ratio of 5:1. The temperature program started at 100℃, increased to 175℃ at a rate of 15℃ / min (held for 5 min), and then increased to 220℃ at a rate of 20℃ / min (held for 2 min). The injection volume was 1 μL, and the injector temperature was 240℃. The concentration of SCFAs in the sample was calculated using a mixed standard solution.

[0105] (3) Analysis of Short-Chain Fatty Acid Content Results: In this example, the content of short-chain fatty acids in the fermentation broth of each culture medium after 48 hours of fermentation was measured, including acetic acid, propionate, and butyrate. Due to the fermentation characteristics of Bifidobacterium, only acetic acid content data was detected. Figure 6 It can be seen that the production of short-chain fatty acids shows a gradual increasing trend over time. Figure 6 c reflects the acetic acid production retention rate in the system, calculated as ((acetic acid production in the bile acid system / acetic acid production in the bile acid-free system) × 100%). In the medium with MIS as the carbon source, acetic acid production consistently remained at a high level above 60%, while it was lower in the Glc control group and the blank control group. This indicates that *Miscanthus sinensis* arabinoxylan can maintain the production of the beneficial metabolite acetic acid in systems containing bile acids. Studies have shown that acetic acid can regulate blood glucose levels and insulin sensitivity. As an important energy source, it can promote intestinal mucosal proliferation and intestinal epithelial cell differentiation, regulate immune cell development, and inhibit inflammation, thereby improving intestinal barrier function. These functions collectively maintain human health and the balance of the intestinal microecology.

[0106] Example 8: Determination of lactic acid during fermentation of Miscanthus arabinoxylan

[0107] (1) Sample processing: The fermentation broth from different time points in Example 6 was centrifuged (6000×g, 10min), and 100μL of the supernatant was collected in a centrifuge tube for testing. A lactic acid assay kit was used. The lactic acid content was detected at 6, 12, 24, 36, and 48 hours during fermentation using BC2230.

[0108] (2) Analysis of lactic acid content results: Figure 7 It can be seen that the content of lactic acid produced by fermentation gradually increases over time. Figure 7 c reflects the lactic acid production retention rate in the system, calculated as ((lactic acid production in the bile acid system / lactic acid production in the bile acid-free system) × 100%). The lactic acid production in the medium with MIS as the carbon source consistently remained at a high level above 70%, while it was at a lower level in the Glc control group and the blank control group. This indicates that Miscanthus arabinoxylan can maintain the production of the beneficial metabolite lactic acid in systems containing bile acids. Studies have shown that lactic acid can promote digestion and nutrient absorption, regulate energy and fat metabolism, and has antioxidant functions, as well as promoting the development of intestinal immune cells.

[0109] Example 9: Determination of pH and total sugar degradation rate during fermentation of Miscanthus arabinoxylan

[0110] (1) Sample processing: The fermentation broth at different time points in Example 6 was centrifuged (6000×g, 10min), and 2mL of supernatant was collected in a centrifuge tube. The pH of the fermentation broth was measured using a FiveEasy Plus FE28 pH meter. Another 50μL of supernatant was collected in a centrifuge tube, and the total carbohydrate content in the fermentation broth was determined using the phenol-sulfuric acid method.

[0111] (2) Determination of total carbohydrates using the phenol-sulfuric acid method: Centrifuge the fermentation broth at 6000×g for 10 min, and take 15 μL of the supernatant to add to 500 μL of distilled water and 500 μL of 3% (w / v) phenol solution. After slowly adding 2 mL of sulfuric acid, shake the mixture vigorously. After standing at room temperature for 30 min, take a 200 μL sample and measure the absorbance at 490 nm. Set the total carbohydrate content of the fermentation broth at 0 h as 100%, and calculate the remaining percentage of total carbohydrates at each time point.

[0112] (3) By Figure 8 It is known that gut microbiota produce a series of metabolites, such as SCFAs, during fermentation, which affect the pH value of the fermentation broth. Changes in pH can, to some extent, reflect the degradation of carbon sources and the growth of bacteria. Figure 8 As shown in a-8b, after 48 hours of fermentation, Glc and Blank lowered the pH to 4.33 and 5.15, respectively, while MIS lowered the pH to 4.98. Under BA stress, Glc and Blank lowered the pH to 5.39 and 5.50, respectively, while MIS lowered the pH to 5.47.

[0113] The remaining total sugar content in the culture medium can reflect the degree of carbohydrate degradation. Figure 8 c reflects the carbohydrate degradation after 48 hours of fermentation without the addition of bile acids, in descending order: Glc (66.8%), MIS (56.1%), and Blank (30.1%). In systems containing bile acids... Figure 8 d. After 48 hours of fermentation, the carbohydrate degradation from highest to lowest was MIS (46.2%), Blank (21.9%), and Glc (20.7%). This suggests that under bile acid stress, arabinoxylan from Miscanthus sinensis may be more likely to resist the stress environment and protect the intestinal flora, while glucose, although a rapid source of carbohydrates, increases bacterial numbers quickly, it does not provide any protective effect.

[0114] Example 10: Determination of fiber morphology changes during fermentation of Miscanthus arabinoxylan

[0115] (1) Sample processing: The fermentation broth at different time points in Example 6 was centrifuged (6000×g, 10min). The supernatant obtained by centrifugation was adjusted to a final ethanol concentration of 70% with anhydrous ethanol. The fermented arabinoxylan was obtained by alcohol precipitation and freeze-dried for subsequent structural characterization analysis.

[0116] (2) Scanning electron microscopy: The conductive adhesive was fixed on the sample holder, and the arabinoxylan powder was evenly sprayed onto the conductive adhesive. Unadsorbed sample was blown away with an air duster. After gold plating at 5kV acceleration voltage, the sample was photographed.

[0117] Figure 9 These are SEM images of Miscanthus arabic xylan before and after fermentation, magnified 5000 times. At the same magnification, the surface structure of Miscanthus arabic xylan differs significantly after fermentation. Before fermentation, the surface layer and cross-section of the raw material are smooth; bacterial fermentation disrupts this structural integrity, resulting in a rough surface and a distinct porous structure. The appearance of this porous structure is delayed in the presence of bile acids, but the number and depth of cracks gradually increase over time. The altered microstructure of Miscanthus arabic xylan holds promise for increasing the specific surface area and porosity of dietary fiber and may potentially influence the adsorption capacity of various compounds through capillary action.

[0118] In summary, according to the embodiments of the present invention, the arabinoxylan prepared in this application has the functions of lowering blood sugar, adsorbing bile acids, and antioxidation in vitro. It can protect the growth vitality of long-chain bifidobacteria in the bile acid environment, maintain the content of beneficial metabolites, and be specifically degraded itself, thereby exerting the effect of beneficial bacteria. It has the potential to be used in combination with probiotics.

[0119] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0120] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Application of Miscanthus arabic in the preparation of products with antioxidant, hypoglycemic or lipid-lowering properties.

2. Application of Miscanthus arabic in the preparation of products that regulate intestinal flora homeostasis.

3. The application as described in claim 2, characterized in that, The product is used to protect the growth viability of Bifidobacterium longum in a bile acid environment.

4. The application as described in claim 2, characterized in that, The product is used to maintain the levels of beneficial metabolites produced by long-chain bifidobacteria.

5. The application as described in claim 4, characterized in that, The beneficial metabolites include short-chain fatty acids and lactic acid.

6. The method for preparing Miscanthus arabic as described in claim 1 or 2, characterized in that, The aforementioned Miscanthus arabinoxylan is prepared from Miscanthus as raw material through alkali treatment, ultrasonic-assisted extraction, water bath heating, alcohol precipitation, and vacuum freeze-drying.

7. The preparation method according to claim 6, characterized in that, The Miscanthus arabinoxylan is prepared by a method including the following steps: The raw material of Miscanthus sinensis is crushed and sieved to obtain Miscanthus sinensis powder; The powdered Miscanthus sinensis was treated with alkali by adding NaOH with a mass-volume concentration of 12% at a material-to-liquid ratio of 1:

20. The alkali-treated sample solution was extracted using an ultrasonic-assisted water bath to obtain the extract. Centrifuge the extract and collect the supernatant; Add ethanol to the supernatant for alcohol precipitation, and adjust the final ethanol concentration to 70%. The alcohol-precipitated solution was centrifuged and the precipitate was collected and freeze-dried under vacuum to obtain the Miscanthus arabinoxylan.

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