Preparation method and use of beet polysaccharide

Abstract

The present invention relates to the technical field of plant polysaccharide extraction, and in particular to a preparation method and use of beet polysaccharide. The beet polysaccharide preparation method provided by the present invention comprises first subjecting beet pulp to acid hydrolysis to obtain a crude extract of beet pectin, then subjecting the crude extract of beet pectin to enzymatic hydrolysis, enriching the neutral sugar side chains acylated with ferulic acid in beet pectin by combining a gel chromatography column and an ion chromatography column, and finally obtaining low-molecular-weight beet polysaccharide by freeze-drying. The beet polysaccharide prepared by the present invention is a low-molecular-weight polysaccharide with good water solubility, which is beneficial for absorption and utilization by organisms; at the same time, the beet polysaccharide prepared by the present invention has a high content of ferulic acid, and exhibits good antioxidant health care effects in animal models, thereby achieving the effect of making full use of beet pulp and improving the added value of beet pulp.

Description

Technical Field

[0001] The invention relates to the technical field of plant polysaccharide extraction, and in particular to a preparation method and application of beet polysaccharide. Background Art

[0002] Beet pulp is a byproduct of the sugar production process. Studies have found that beet pulp contains 80% dietary fiber, specifically 20% cellulose, 25% hemicellulose, 25% pectin and a small amount of protein, lignin, and ash. Dietary fiber is a class of ingredients containing polysaccharides, oligosaccharides, lignin, inulin, resistant starch and plant matrix. It can be divided into water-soluble dietary fiber and insoluble dietary fiber according to different water-soluble characteristics. Water-soluble dietary fiber has a wide range of physiological effects, such as adjusting sugar and lipid metabolism, reducing human cholesterol content, preventing cardiovascular and cerebrovascular diseases, and preventing constipation. Therefore, current research on beet pulp mainly focuses on the extraction and modification of water-soluble dietary fiber.

[0003] Gong Ran published a paper titled "Research on the Extraction and Modification of Dietary Fiber from Beet Waste", which used microwave extraction and enzymatic extraction to purify beet dietary fiber, and modified it by carboxymethylation to increase its water-soluble dietary fiber content and total dietary fiber content. By comparing with traditional chemical extraction methods, the process parameters and process flow for comprehensive utilization of dry beet waste were determined. Finally, the paper used carboxymethylation to modify the beet dietary fiber to a weight ratio of 36.51% in beet waste, which can effectively improve the extraction rate of dietary fiber in beet waste.

[0004] Patent document CN110419744A discloses a method for extracting dietary fiber from beet pulp, which mainly solves the technical problem of low dietary fiber extraction rate in beet pulp in the existing method. The extraction method is to put the remaining beet pulp after sugar production into water and immediately heat it to above 90°C to destroy the enzymes therein and prevent the enzymatic reaction of the beet pulp, then grind the beet pulp with a colloid mill, and further purify it with a physical separation method to obtain beet dietary fiber with higher purity. The extraction method uses beet pulp as a raw material to prepare dietary fiber, has high economic benefits, and the production process is very safe, so that the dietary fiber powder has high edible safety, and uses beet pulp as a raw material to make it rich in nutrients.

[0005] Patent document CN104886547A discloses a beet dietary fiber and a preparation method thereof. The extraction method adopts a decolorization pretreatment-drying-ultrafine grinding method, which further optimizes the color, dietary fiber content, water holding capacity and oil holding capacity of the extracted beet dietary fiber powder. It is particularly suitable for the use of low-cost dietary fiber supplementation in the food industry, thereby improving the economic benefits of the sugar industry and providing a better way for the further comprehensive utilization of waste.

[0006] Beet pectin is an important water-soluble dietary fiber, a polysaccharide with multiple physiological functions and unique physical and chemical properties, which has preventive and inhibitory effects on obesity, diabetes, cardiovascular disease, intestinal cancer and other diseases. However, there is little in-depth research on beet pectin, and natural beet pectin has the characteristics of large molecular weight and poor water solubility, which is not conducive to the absorption and utilization of organisms and cannot fully exert its biological activity, thus limiting the application of beet pectin. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation method and application of beet polysaccharide to increase the added value of beet pulp. The preparation method of beet polysaccharide provided by the present invention is to firstly acid hydrolyze beet pulp to obtain a crude extract of beet pectin, then enzymatically hydrolyze the crude extract of beet pectin, enrich the neutral sugar side chains acylated with ferulic acid in beet pectin by combining a gel chromatography column and an ion chromatography column, and finally obtain low molecular weight beet polysaccharide by freeze drying.

[0008] The method for preparing beet polysaccharide provided by the present invention comprises the following steps:

[0009] Step S1. crushing the dried beet pulp to obtain beet pulp powder, uniformly dispersing the beet pulp powder in distilled water to obtain a raw material liquid, adjusting the pH value of the raw material liquid, stirring at 70-90° C. for 1-2 hours, cooling to room temperature, filtering, centrifuging at 6000-10000 g for 18-22 minutes, collecting the supernatant, fully mixing the supernatant with anhydrous ethanol, centrifuging at 6000-10000 g for 20 minutes, collecting the precipitate, washing the precipitate with 95% anhydrous ethanol for 2-4 times, and drying at 40-50° C. for 24 hours to obtain a crude extract of beet pectin;

[0010] Step S2. dissolving the crude extract of beet pectin obtained in step S1 in distilled water to obtain a crude polysaccharide solution, adding pectinase to the crude polysaccharide solution, adjusting the pH of the crude polysaccharide solution to 3.5, performing enzymolysis at 45-55° C. for 24 hours, then heating in boiling water for 4-6 minutes, centrifuging at a speed of 5000 rpm for 4-5 minutes, collecting the supernatant, then adding trypsin to the supernatant, adjusting the pH of the supernatant to 7, performing enzymolysis at 35-40° C. for 12 hours, inactivating the enzyme, and centrifuging at a speed of 5000 rpm for 5 minutes to obtain a deproteinized solution;

[0011] Step S3. Apply the deproteinized solution obtained in step S2 to a DEAE-52 agarose column, and gradually elute with distilled water, 0.2 mol / L and 0.4 mol / L sodium chloride, collect the eluate with 0.4 mol / L sodium chloride, further purify on Sephadex G-100, elute with distilled water, detect the polysaccharide content, collect the main peak components, and lyophilize to obtain.

[0012] Furthermore, in step S1, the material-liquid ratio of beet pulp powder to distilled water is 1 g:25 mL.

[0013] Furthermore, in step S1, the pH of the raw material solution is adjusted to 1-2.

[0014] Furthermore, in step S1, the mixing volume ratio of the supernatant and anhydrous ethanol is 1:3, and the mixture is mixed at 4° C. for 12 hours.

[0015] Furthermore, in step S2, the solid-liquid ratio of the crude beet pectin extract to distilled water is 10 mg:1 mL.

[0016] Furthermore, in step S2, the amount of pectinase added is 0.2% (m / v) of the volume of the crude polysaccharide solution, and the amount of trypsin added is 0.2% (m / v) of the volume of the crude polysaccharide solution.

[0017] Furthermore, the specification of the DEAE-52 agarose column in step S3 is 3.5×40 cm, and the elution rate is 1 mL / min.

[0018] Furthermore, in step S3, the specification of Sephadex G-100 is 2.4×40 cm, and the elution rate is 0.5 mL / min.

[0019] In addition, the present invention also provides the use of the beet polysaccharide prepared by the beet polysaccharide preparation method in the preparation of antioxidant products.

[0020] The substances that play a functional role in the crude extract of beet pectin are very limited. The inventors have found that beet pectin has a high neutral sugar side chain. The RG-I region in pectin is mainly composed of neutral sugar side chains, which is called the "hairy region". The side chains are mainly composed of neutral sugars, including galactose, arabinose, glucose and other sugars, such as focusing sugar, mannose and xylose. The ferulic acid attached to the neutral sugar side chain of beet pectin is a key structure that affects its antioxidant activity. As a natural polyphenol compound, ferulic acid plays an antioxidant physiological function by removing free radicals in the body. Therefore, how to increase the content of ferulic acid in beet pectin is the key to improving the antioxidant effect of beet pectin.

[0021] The inventor has obtained a method for enriching the content of ferulic acid in beet pectin through a large number of exploratory experiments, which is specifically: firstly, beet pulp is subjected to acid hydrolysis to obtain a crude extract of beet pectin, then the crude extract of beet pectin is subjected to enzymatic hydrolysis, and neutral sugar side chains acylated with ferulic acid in beet pectin are enriched by combining a gel chromatography column and an ion chromatography column, and finally low-molecular-weight beet polysaccharides are obtained by freeze drying. The mass fraction of ferulic acid in the low-molecular-weight beet polysaccharide prepared by the present invention is 22.5 μg / mg, and the average molecular weight is 33.31 KDa. The low-molecular-weight beet polysaccharide is an acidic polysaccharide, which is a heteropolysaccharide composed of fucose, rhamnose, arabinose, galactose, glucose, galacturonic acid and glucuronic acid in a molar ratio of 0.46:51.87:1.85:14.94:0.36:26.05:4.47.

[0022] The inventors have found through experiments that the low molecular weight beet polysaccharide prepared by the present invention has a significant antioxidant effect. Specifically, the low molecular weight beet polysaccharide can improve the survival rate of nematodes damaged by paraquat, gradually eliminate the occurrence of accelerated accumulation of lipofuscin induced by paraquat, reduce the level of ROS in the nematodes, and enhance the antioxidant capacity of the nematodes. At the same time, the low molecular weight beet polysaccharide also has a protective effect on the antioxidant stress of antioxidant enzymes, and can also reduce the degree of damage to lipid peroxidation cells in the nematodes, and has a significant antioxidant effect.

[0023] In summary, compared with the prior art, the method for preparing beet polysaccharide provided by the present invention has the following advantages:

[0024] (1) The beet polysaccharide prepared by the beet polysaccharide preparation method provided by the present invention has low molecular weight and good water solubility, which is conducive to the absorption and utilization of organisms. It is a low molecular weight beet pectin polysaccharide with good water solubility;

[0025] (2) The beet polysaccharide preparation method provided by the present invention has a high content of ferulic acid in the beet polysaccharide, which can effectively improve the antioxidant effect of the beet polysaccharide, ensure the health care effect of the beet polysaccharide, and fully utilize the beet pulp and increase the added value of the beet pulp. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a process flow chart for the extraction and purification of beet polysaccharides in Example 1.

[0027] Figure 2 is the purification elution profile, where: Figure 2 -A is the elution spectrum of crude SBP on DEAE-52 cellulose column, Figure 2 -B is the UV spectrum of the eluted component at 320 nm, Figure 2 -C is the elution profile of SBP on Sephadex G-100 column, Figure 2 -D is the HPGPC spectrum of SBP-3A.

[0028] Figure 3 Figure 2 is the monosaccharide composition diagram of SBP-3A, where: Figure 3 -A is the monosaccharide composition of SBP-3A (1: caramel, 2: rhamnose, 3: arabinose, 4: galactose, 5: glucose, 6: xylose, 7: mannose, 8: fructose, 9: ribose, 10: galacturonic acid, 11: glucuronic acid, 12: glucuronic acid, 13: mannuronic acid), Figure 3 -B is the FT-IR spectrum of SBP-3A, Figure 3 -C is the HPLC spectrum of ferulic acid standard and SBP-3A at 320 nm.

[0029] Figure 4 is the nuclear magnetic resonance spectrum of SBP-3A, wherein: Figure 4 -A is 1H NMR spectrum, Figure 4 -B is the 13C NMR spectrum, Figure 4 -C is the HH COSY spectrum, Figure 4 -D is the HSQC spectrum, Figure 4 -E is the HMBC spectrum, Figure 4 -F is NOESY spectrum.

[0030] Figure 5 This is the scanning electron microscope observation image of SBP-3A.

[0031] Figure 6 This is an atomic force microscope observation image of SBP-3A.

[0032] Figure 7 Figure 2 is a diagram showing the protective effect of SBP-3A on oxidative stress in paraquat-attacked nematodes, where: Figure 7 -A is the survival diagram of SBP-3A against paraquat-challenged nematodes, Figure 7 -B is the generated graph of ROS, Figure 7 -C is the intensity effect of paraquat attack on nematodes on lipofuscin, Figure 7 -D are representative fluorescence micrographs of lipofuscin accumulation.

[0033] Figure 8 This is a graph showing the effects of SBP-3A on paraquat-induced oxidative stress in nematodes (NO production, SOD, CAT and GSH antioxidant enzyme activities, and MDA levels).

[0034] Fig. 9 Graph showing daily progeny production and total number per worm during the adult reproduction stage. DETAILED DESCRIPTION

[0035] The present invention is further described below by specific examples, and the present invention is not limited to the following examples. Within the scope of the present invention or without departing from the content, spirit and scope of the present invention, the changes, combinations or substitutions made to the present invention are obvious to those skilled in the art and are included in the scope of the present invention. All materials and reagents of the present invention are commercially available and can be purchased.

[0036] Example 1. Preparation method of beet polysaccharide

[0037] Step S1. crushing the dried beet pulp to obtain beet pulp powder, and uniformly dispersing the beet pulp powder in distilled water, wherein the solid-liquid ratio of the beet pulp powder to distilled water is 1 g:25 mL, to obtain a raw material liquid, adjusting the pH value of the raw material liquid to 1.5, stirring at 80° C. for 1 hour, cooling to room temperature, filtering through a 400-mesh filter cloth, centrifuging at 6000 g for 20 minutes, collecting the supernatant, mixing the supernatant with anhydrous ethanol at a volume ratio of 1:3 at 4° C. for 12 hours, centrifuging at 10000 g for 20 minutes, collecting the precipitate, washing the precipitate with 95% anhydrous ethanol for 3 times, and drying at 45° C. for 24 hours to obtain a crude extract of beet pectin;

[0038] Step S2. dissolving the crude beet pectin extract obtained in step S1 in distilled water, wherein the solid-liquid ratio of the crude beet pectin extract to distilled water is 10 mg:1 mL, to obtain a crude polysaccharide solution, adding pectinase to the crude polysaccharide solution, wherein the amount of pectinase added is 0.2% (m / v) of the volume of the crude polysaccharide solution, adjusting the pH of the crude polysaccharide solution to 3.5, performing enzymolysis at 50° C. for 24 h, then heating in boiling water for 5 min, centrifuging at a speed of 5000 rpm for 5 min, collecting the supernatant, then adding trypsin to the supernatant, wherein the amount of trypsin added is 0.2% (m / v) of the volume of the crude polysaccharide solution, adjusting the pH of the supernatant to 7, performing enzymolysis at 37° C. for 12 h, inactivating the enzyme, and centrifuging at a speed of 5000 rpm for 5 min to obtain a deproteinized solution;

[0039] Step S3. Apply the deproteinized solution obtained in step S2 to a DEAE-52 agarose column with a specification of 3.5×40 cm, and gradually elute with distilled water, 0.2 mol / L and 0.4 mol / L sodium chloride at a rate of 1 mL / min, collect the eluate of 0.4 mol / L sodium chloride, and further purify it on a Sephadex G-100 with a specification of 2.4×40 cm, elute with distilled water at a rate of 0.5 mL / min, detect the polysaccharide content, collect the main peak components, and lyophilize to obtain beet polysaccharide (SBP-3A).

[0040] Example 2, Preparation method of beet polysaccharide

[0041] Step S1. The dried beet pulp is crushed to obtain beet pulp powder, and the beet pulp powder is evenly dispersed in distilled water, wherein the solid-liquid ratio of the beet pulp powder to distilled water is 1 g:25 mL, to obtain a raw material liquid, the pH value of the raw material liquid is adjusted to 1.8, and the mixture is stirred at 75° C. for 2 h. After cooling to room temperature, the mixture is filtered through a 400-mesh filter cloth, centrifuged at 8000 g for 22 min, and the supernatant is collected. The supernatant is mixed with anhydrous ethanol at a volume ratio of 1:3 at 4° C. for 12 h, and centrifuged at 10000 g for 20 min. The precipitate is collected, and the precipitate is washed 4 times with 95% anhydrous ethanol, and dried at 50° C. for 24 h to obtain a crude extract of beet pectin;

[0042] Step S2. dissolving the crude beet pectin extract obtained in step S1 in distilled water, wherein the solid-liquid ratio of the crude beet pectin extract to distilled water is 10 mg:1 mL, to obtain a crude polysaccharide solution, adding pectinase to the crude polysaccharide solution, wherein the amount of pectinase added is 0.2% (m / v) of the volume of the crude polysaccharide solution, adjusting the pH of the crude polysaccharide solution to 3.5, performing enzymolysis at 48° C. for 24 h, then heating in boiling water for 4 min, centrifuging at a speed of 5000 rpm for 4 min, collecting the supernatant, then adding trypsin to the supernatant, wherein the amount of trypsin added is 0.2% (m / v) of the volume of the crude polysaccharide solution, adjusting the pH of the supernatant to 7, performing enzymolysis at 35° C. for 12 h, inactivating the enzyme, and centrifuging at a speed of 5000 rpm for 5 min to obtain a deproteinized solution;

[0043] Step S3. Apply the deproteinized solution obtained in step S2 to a DEAE-52 agarose column with a specification of 3.5×40 cm, and gradually elute with distilled water, 0.2 mol / L and 0.4 mol / L sodium chloride at a rate of 1 mL / min, collect the eluate of 0.4 mol / L sodium chloride, and further purify it on a Sephadex G-100 with a specification of 2.4×40 cm, elute with distilled water at a rate of 0.5 mL / min, detect the polysaccharide content, collect the main peak components, and lyophilize to obtain beet polysaccharide (SBP-3A).

[0044] Experimental Example 1: Structural Characteristics Analysis Experiment of Beet Polysaccharide

[0045] 1. Experimental materials:

[0046] The sugar beet polysaccharide (SBP-3A) prepared in Example 1, DEAE-cellulose-52 were purchased from Rhawn, Sephadex G-100 was purchased from Shanghai Yuanye, and all other chemical reagents were of analytical or chromatographic grade.

[0047] 2. Experimental methods:

[0048] The structural characteristics of the beet polysaccharide obtained in Example 1 were determined, wherein:

[0049] 2.1. Quantitative determination of ferulic acid:

[0050] The ferulic acid content in each component of beet pectin polysaccharide was quantified by HPLC. A 2 mg / mL sample of SBP-3A prepared in Example 1 was dissolved in 70% methanol, 0.1% sodium hydroxide solution was added, and ultrasonic treatment was performed for 30 min. After filtration through a 0.22 μm membrane, the sample was tested on an HPLC machine (HPLC1260, Agilent Technology, USA). The operating conditions of HPLC were as follows: Agilent extended 18 column (mm×mm), methanol-0.1% glacial acetic acid mobile phase (70:30, v / v); detection wavelength: 320 nm; flow rate 0.5 mL / min, injection volume 10 μL, column temperature 30°C, and analysis time 15 min.

[0051] 2.2 Molecular weight determination:

[0052] The molecular weight of SBP-3A prepared in Example 1 was determined by high performance gel permeation chromatography and differential refractometer. Separation was performed on a BRT105-103-101 gel column (8×300 mm) and eluted with a 0.2 mol / L sodium chloride solution at a flow rate of 0.8 mL / min. A 5 mg / mL sample of SBP-3A polysaccharide prepared in Example 1 was dissolved in the mobile phase and filtered through a 0.22 μm membrane filter before injection. The molecular weight of beet pectin polysaccharides (dextran 7380, 21950, 44520, 109900, 410200, 755900 Da) was determined using dextran standards.

[0053] 2.3. Monosaccharide composition analysis:

[0054] The monosaccharide composition of SBP-3A was determined by ion chromatography (ICS 5000+, Thermo Fisher Scientific, USA) equipped with an electrochemical detector and Dionex TM CarboPac TMPA20 (150 × 3.0 × 3.0 mm, 10 μm), SBP-3A polysaccharide sample (5 mg) was hydrolyzed with 1 mL 2 mol / L trifluoroacetic acid solution (TFA) at 121 °C for 2 h. After hydrolysis, the excess TFA was removed under nitrogen blowing. Then it was washed with anhydrous methanol three times. The hydrolyzed sample was then dissolved in deionized water and injected into the column for ICS analysis. The chromatographic conditions were: mobile phase, A (water), B (0.1 mol / L sodium hydroxide), C (0.1 mol / L sodium hydroxide, 0.2 mol / L NaAc), flow rate 0.5 ml / min, column temperature 30°C; elution gradient: 0 min (95:5:0, V / V), 26 min (85:5:10, V / V), 42 min (85:5:10, V / V), 42.1 min (60:0:40, V / V), 52 min (60:40:0, V / V), 52.1 min (95:5:0, V / V), 60 min A / B / C (95:5:0, V / V).

[0055] 2.4、Infrared analysis:

[0056] Before FT-IR analysis, dry potassium bromide was mixed with SBP-3A in a ratio of 150:1 and pressed into thin sheets. The FT-IR spectrum of SBP-3A was obtained at 400 cm -1 ~4000cm -1 Collect between.

[0057] 2.5. Methylation analysis:

[0058] The uronic acid of polysaccharide was reduced by methylation. 4 Restore the sample, dialyze the lyophilized solution, and use CH 3 I was methylated in NaOH / DMSO. The methylation process was repeated twice to ensure the completeness of the methylation. After the methylation was completed, the overmethylated product was hydrolyzed with 2 mol / L TFA at 121 °C for 1.5 h and then with NaBD 4 After reduction, acetylation with acetic anhydride was performed for 2.5 h (100 °C). Finally, the acetate derivatives were analyzed by gas chromatography-mass spectrometry (Agilent Technologies, USA).

[0059] 2.6. Nuclear Magnetic Resonance Analysis:

[0060] 40 mg of the SBP-3A sample prepared in Example 1 was dissolved in 1 mL of D2O, and 1HNMR, HSQC, 1H-1HCOSY and HMBC spectra were recorded on a 500 MHz NMR instrument.

[0061] 2.7. Surface microscopic analysis:

[0062] Part of the SBP-3A sample prepared in Example 1 was dispersed in an ultrapure water solution and subjected to ultrasound for 5 minutes, and then a few drops of the dispersed liquid were dripped onto a mica sheet, dried at room temperature for 30 minutes, and the sample was photographed using a Bruker Dimension IconAFM instrument. A trace amount of sample was directly adhered to the conductive adhesive, and gold was sprayed for 45 seconds using a Quorum SC7620 sputtering coater at 10 mA; then the sample morphology was photographed using a ZEISS GeminiSEM 300 scanning electron microscope, with an accelerating voltage of 3 kV and a SE2 secondary electron detector as the detector.

[0063] 3. Experimental results:

[0064] 3.1. Separation and purification results:

[0065] The separation and purification results are as follows Figure 1 and Figure 2 shown. Figure 1 This is a process flow chart of extraction and purification of beet polysaccharide obtained in Example 1; Figure 2 is the purification elution profile, where: Figure 2 -A is the elution spectrum of crude SBP on DEAE-52 cellulose column, Figure 2 -B is the UV spectrum of the eluted component at 320 nm, Figure 2 -C is the elution profile of SBP on Sephadex G-100 column, Figure 2 -D is the HPGPC spectrum of SBP-3A.

[0066] like Figure 2 -A, DEAE-52 cellulose column (SBP-1, SBP-2 and SBP-3) was used to separate the beet pectinase solution into three components. The ferulic acid in SBP-3A was qualitatively analyzed by using an ultraviolet detector, and the signal of each component at 320nm was detected. The component containing ferulic acid fragments was ferulic acid acylated beet pectin neutral sugar side chain. The ultraviolet spectrum of the eluted component at 320nm was as follows Figure 2 -B. From Figure 3 As can be seen from the figure, the content of ferulic acid is the highest in the 0.4 mol / L sodium chloride elution fraction. The SBP-3 containing the ferulic acid fraction was further purified using a Sephadex G-100 column. The elution profile on the Sephadex G-100 column is shown in Figure 2. Figure 2 -C, a homogeneous polysaccharide SBP-3A was obtained.

[0067] 3.2. Quantitative analysis results of ferulic acid:

[0068] The content of ferulic acid in the neutral sugar side chain of beet pectin was determined by HPLC. Figure 3As shown in the chromatogram, the peaks of ferulic acid and other components were well separated. The regression equation for the determination of ferulic acid content was established: y = 68.516x-2.5656 (R2 = 0.9998), 1.25-80 μg / mL. The mass fraction of ferulic acid in SBP-3A was 22.5 μg mg -1 .

[0069] 3.3. Molecular weight determination results:

[0070] The average molecular weight (Mw) of SBP-3A is shown in Figure 2 -D. The Mw distribution of SBP-3A was determined by high performance gel permeation chromatography. Figure 2 As shown in Figure 2, the HPGPC spectrum of SBP-3A is symmetrical and stable at the baseline, with a polydispersity index of 1.02, indicating that SBP-3A is a homogeneous polysaccharide after separation and purification by chromatographic column and gel column. A standard curve was established using dextran of different relative molecular weights as standard products. y = -0.204x + 10.99 (R 2 =0.999). The average molecular weight of SBP-3A is 33.31 KDa.

[0071] 3.4. Monosaccharide composition analysis results:

[0072] The monosaccharide composition of SBP-3A was determined. The properties of the monosaccharides in the polysaccharide samples were determined by comparison with the standard. Figure 3 As shown, where: Figure 3 -A is the monosaccharide composition of SBP-3A (1: caramel, 2: rhamnose, 3: arabinose, 4: galactose, 5: glucose, 6: xylose, 7: mannose, 8: fructose, 9: ribose, 10: galacturonic acid, 11: glucuronic acid, 12: glucuronic acid, 13: mannuronic acid), Figure 3 -B is the FT-IR spectrum of SBP-3A, Figure 3 -C is the HPLC spectrum of ferulic acid standard and SBP-3A at 320 nm.

[0073] from Figure 3 It can be seen that SBP-3A is a heteropolysaccharide, which is composed of fucose, rhamnose, arabinose, galactose, Glc, galacturonic acid, and glucuronic acid in a molar ratio of 0.46:51.87:1.85:14.94:0.36:26.05:4.47. In addition, the content of uronic acid indicates that SBP-3A is an acidic polysaccharide.

[0074] 3.5. Infrared analysis results:

[0075] The infrared spectrum of SBP-3A shows several typical polysaccharide absorption bands. The absorption band of SBP-3A polysaccharide is 3427.43 cm -1 The characteristic absorption peak at 2927.88cm is the strong OH stretching vibration. -1 and 1417.65cm -1 The absorption peaks at 1041.54 cm are due to CH stretching and CH bending vibrations. -1 The absorption peak at 1251.98 cm-1 of SBP-3A confirms the existence of COH stretching vibration of the pyranose ring. -1 The absorption peak at 894.95 cm-1 is caused by the vibration of -OH (carboxyl) group, indicating that SBP-3A contains uronic acid. -1 The absorption peak at

[0076] 3.6. Methylation and NMR analysis results:

[0077] Based on the CCRC spectral database of PMAAs, the results are shown in Table 1. SBP-3A mainly contains eight types of glycosidic bonds, t-Rhap, 1,2-linked-Rhap, t-Galp, 1,2,4-linked-Rhap, 1,4-linked-GalpA, 1,4-linked-Glcp, 1,6-linked-Galp, 1,3,4-linked-Galp with molar percentages of 2.64: 44.21: 6.93: 4.69: 33.87: 0.81: 2.26: 4.58, respectively. These results indicate that the main chain of SBP-3A contains 1,2-linked-Rhap and 1,4-linked-GalpA, which is consistent with the monosaccharide composition. However, other monosaccharides such as arabinose and glucose were not detected due to their low content.

[0078] Table 1

[0079] Table 1

[0080] MethylationAnalysis of SBP-3A.

[0081]

[0082] NMR provides detailed structural information of carbohydrates, including α- or β-isomer configuration, connection pattern, and sugar unit sequence. The structural characteristics of SBP-3A were studied using 1H NMR and 13C NMR spectra. Figure 4 As shown:

[0083] By analyzing the anomeric hydrogen and anomeric carbon of polysaccharide molecules in one-dimensional 1H NMR and 13C NMR spectra, the specific configuration of its glycosidic bond and the type of monosaccharide composition can be determined. Generally speaking, the chemical shift of anomeric hydrogen of α-configuration polysaccharide molecules is δ>5.0, and the chemical shift of anomeric hydrogen of β-configuration polysaccharide molecules is δ<5.0. The chemical shift of anomeric hydrogen is located at 4.5-5.50ppm, indicating that α- and β-configuration glycosidic bonds exist in the polysaccharide molecules at the same time. The 1H NMR spectrum has a clear peak at the high field (δ1.17), which is attributed to the H on the Rhap methyl group. In the 13C NMR spectrum, the carbon signal of δ174.9 is the carboxyl carbon of GalpA. These nuclear magnetic resonance evidences are consistent with the rich rhamnose (51.87%) and galacturonic acid (26.05%) in the monosaccharide composition. In the anomeric region, the signals at 5.21 ppm and 4.60 ppm were assigned to H-1 of rhamnose and H-1 of galactose, respectively, and 4.96 ppm was assigned to H1 of galacturonic acid.

[0084] According to the HSQC spectrum, the following cross signal peaks were obtained: H 5.21 / C 98.3, H 4.60 / C 103.5, H 4.96 / C 97.4, H5.01 / C 100.3, H 4.56 / C 98.3, H 4.54 / C 96.1 ppm, indicating that there are 5 sugar residues in SBP-3A, which are named as A, B, C, D, E, and F. For sugar residue A: at δH 5.21 (H1) / δH 4.06 (H2), δH 4.06 / δH 3.82 (H3), δH3.83 / δH 3.35 (H4), δH 3.35 / δH4.13 and δH 4.13 / δH Some paired 1H-1H related signals were clearly observed between 1.18 and 1.18. These five proton signals from the same spin system were easily identified as H1 to H5 of →2)-α-Rhap-(1→. According to HSQC, the corresponding chemical shifts of C1-C5 were δ98.3, 76.3, 71.9, 71.0, 74.1, and 16.5 ppm. Combined with the monosaccharide composition, methylation results and previous literature reports, it was inferred that the A sugar residue was →2)-α-Rhap-(1→. In the COSY spectrum, at 4.96δH (abnormal proton) / δH 3.82(H2), δH 3.82 / δH 4.06(H3), δH 4.06 / δH4.36(H4) and δH4.36 / δH Some paired 1H-1H related signals were clearly observed between 4.60 (H5). These five proton signals from the same spin system can be easily identified as H1 to H5 of →4)-α-GalpA-(1→(residue C). These two residues are characteristic repeating units that often appear in RG-I pectin. Similarly, the chemical shifts of sugar residues B, D, and E can be obtained based on 1H-1H COSY and HSQC, as shown in Table 2.

[0085] Table 2

[0086] Table 2

[0087] 1H / 13CNMR chemical shift assignments ofSBP-3A.

[0088]

[0089] According to the monosaccharide composition, methylation results and related literature reports, it can be inferred that sugar residue B is: →6)-β-Galp-(1→, sugar residue D is: 2,4)-α-Rhap-(1→, and sugar residue E is: Galp-(1→. Based on the above results, the comprehensive assignment of all glycosidic bonds is listed in Table 2.

[0090] The connection between H1 of →2)-α-Rhap-(1→(residue A) and H-4 of →4)-α-Galp A-(1→(residue C) was easily identified in the NOE spectrum (δ 5.21 / 4.36 ppm). In addition, the connection between H1 of A and H1, H3, and H5 of C was identified as →2)-α-Rhap-(1→(residue A) and →4)-α-Galp A-(1→(residue C) The connection pattern at the O-4 position of A-(1→). This was further confirmed by the correlation of H1 of A with C4 of C at δ 5.21 / 77.03 ppm in HMBC. The same method was used to determine the connection of the O-2 position of →4)-α-GalpA-(1→) and →2)-α-Rhap-(1→). The repeating unit was established as [→2)-α-Rhap-(1→4)-α-GalpA-(1→2)-α-Rhap-(1→], which is commonly found in the RG-I part of acidic heteropolysaccharides.

[0091] In HMBC, at δ5.21 / 69.0ppm, the correlation between H1 of residue A and C4 of residue D indicated the existence of a linked structure →2)-α-Rhap-(1→2,4)-α-Rhap-(1→. At the same time, in the NOE spectrum, H-1 of residue D and H-2 of residue A had related signal peaks, which also confirmed this. The cross peak at δ4.6 / 103.5ppm corresponds to H1 / C1 of →6)-β-Galp-(1→(residue B). In the NOE spectrum, In the spectrum, H1 of residue B and H2, H3, and H5 of residue D have related signal peaks, indicating the existence of a chain structure →6)-α-Galp-(1→2,4)-α-Rhap-(1→. However, due to the low content of glucose, its carbon NMR signal is too weak to be detected. In general, the results show that the possible structure of SBP-3A is based on RG-I as the main chain, with a →6)-β-Galp-(1→ branch at the O-4 position of Rha. The possible structure is shown in Formula I:

[0092]

[0093] 3.7. AFM and SEM measurement results:

[0094] The results of SEM observation of SBP-3A are shown in Figure 5 The surface morphology images of SBP-3A were enlarged by 2000× and 20000×, respectively. The surface morphology was irregular block-like with some bead-like structures, and the surface was smooth with some cracks and multi-suture structures, which may be the reason why it easily absorbs water and re-dissolves.

[0095] The atomic force microscopy results of SBP-3A are shown in Figure 6As shown. The SBP-3A molecule is not a linear structure, but a compact structure (spherical particle, called "a"), with a large number of protrusions in the center of the polysaccharide surface, indicating that the SBP-3A branches are intertwined to form a polymer. Its compact structure is formed by multi-molecular aggregation, and the vertical height of small objects is estimated to be 4-5nm, and the vertical height of large objects is estimated to be 10nm. Most molecules exist in the form of aggregates. These aggregates are formed by the interaction and mixing of multiple polymers and molecules during the drying process.

[0096] Experimental Example 2: Determination of the Antioxidant Activity of Beet Polysaccharides in Vitro

[0097] 1. Experimental materials:

[0098] Beet polysaccharide (SBP-3A) prepared in Example 1.

[0099] 2. Experimental methods:

[0100] 2.1、C. elegans culture:

[0101] Wild-type C. elegans strain (N2) and uracil auxotrophic Escherichia coli strain OP50 were used. Nematodes were grown and maintained on standard laboratory nematode growth medium (NGM) agar plates at 20°C. NGM was supplied with a suspension of E. coli OP50. Age-synchronized populations of first-stage larvae (L1) were obtained from gravid hermaphrodites by sodium hydroxide and hypochlorous acid, and then centrifugation-purified eggs were incubated in M9 buffer overnight. Synchronized populations of L4 nematodes were obtained 2 days after L1 synchronization.

[0102] 2.2 Experimental design

[0103] To evaluate the protective potential of SBP-3A against paraquat-induced oxidative stress in nematodes, synchronized L4 nematodes were randomly divided into 4 groups according to the treatment of SBP and / or paraquat, and 4 culture dishes were designed for each group and cultured at 20°C for 5 days. SBP-3A was dissolved in an inactivated Escherichia coli OP50 suspension to obtain 0, 2, and 4 mg / mL culture fluids as nutrient supply, and the nematodes were transferred to fresh culture dishes every day. 40 mmol / L paraquat was added to both the model group and the drug-treated group to induce oxidative stress in the nematodes.

[0104] 2.3. Survival rate determination:

[0105] The survival rate of the nematodes was assessed at the treatment endpoint. There were approximately 30 nematodes per plate per group, for a total of 100 to 130 nematodes per group. Nematodes that did not respond to repeated mild mechanical stimulation were declared dead and removed from the plate. The survival rate of the nematodes in each group was calculated.

[0106] 2.4. Lipofuscin determination:

[0107] Randomly selected nematodes (about 8 per plate) were anesthetized with 10 mmol / L imidazole hydrochloride, mounted on 2% agar, and observed in bright and dark fields using a fluorescence microscope, and then photographed. Relative fluorescence was quantified using ImageJ software.

[0108] 2.5 ROS determination:

[0109] After 5 days of incubation with SBP-3A, nematodes were collected in M9 buffer. The nematodes were added to 200 μL of lysis buffer and incubated at 4 °C for 1 h to separate the tissues. The supernatant was added to a 96-well black plate (50 μL / well) after homogenization and centrifugation using a tissue grinder, and DCFH-DA solution (50 μL, 100 μmol / L) was added to each well. The fluorescence intensity of the worms was measured using a microplate reader (SpectraMax i3x, CA, USA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

[0110] 2.6. Determination of oxidative stress and antioxidant biomarkers:

[0111] Three 60 mm culture dishes were designed for each group (~5,000 larvae were placed in one dish, ~15,000 larvae per group). C. elegans were added with 200 μL of ripe lysis buffer and incubated at 4°C for 1 hour to separate tissues. Homogenize with tissue hemolytic agent. After centrifugation, the supernatant was removed, the mixture was centrifuged at 3000 r / min for 10 min at 4°C, and the upper aqueous layer was transferred to a new EP tube for enzymatic assay. The levels of NO and MDA (as oxidative damage markers) and the activity levels of SOD, CAT, and GSH (as antioxidant markers) were determined using commercial kits according to the manufacturer's instructions. The protein content was determined by the bichondronic acid method (BCA method) using bovine serum albumin as a standard, and the nematodes were taken for endpoint measurement to evaluate the physiological status related to oxidative stress.

[0112] 2.7. Determination of egg laying number:

[0113] The synchronized N2 nematodes were cultured to the L4 stage, and the experimental dishes were prepared: NGM culture dishes containing OP50 bacterial solution with three concentrations of SBP-3A 0mg / mL, 2mg / mL, and 4mg / mL. Three nematodes were randomly selected from each group and placed in three 35mm culture dishes. They were transferred to fresh culture dishes every day and allowed to lay embryos. The number of eggs laid by each nematode and the total number of eggs laid by each group of nematodes were recorded every day.

[0114] 3. Experimental results:

[0115] The experimental results are as follows Figure 7 to Figure 9 As shown: Figure 7 The protective effect of SBP-3A on oxidative stress in paraquat-challenged nematodes: Figure 7 -A is the survival diagram of nematodes attacked by paraquat by SBP-3A, Figure 7 -B is the generated graph of ROS, Figure 7 -C is the intensity effect of paraquat attack on nematodes on lipofuscin, Figure 7 -D is a representative fluorescence micrograph of lipofuscin accumulation. Data are presented as the mean (n=3) ± SEM of three independent experiments. Figure 8 This is the effect of SBP-3A on paraquat-induced oxidative stress in nematodes (NO production, SOD, CAT and GSH antioxidant enzyme activities and MDA level). Fig. 9 Graph showing daily progeny production and total number per worm during the adult reproduction stage.

[0116] 3.1. Survival rate determination results:

[0117] Paraquat (PQ) can cause oxidative stress as a trigger for the production of ROS. Therefore, paraquat was used as a model group to study the effects of SBP-3A on the antioxidant defense system of Caenorhabditis elegans. In this experiment, synchronized L4 nematodes were cultured on OP50 bacterial culture plates with different substances added. The blank control group (control) was only coated with OP50 bacterial culture, the model group was OP50 bacterial culture containing 40mmol / L paraquat, and the polysaccharide group was OP50 bacterial culture containing 40mmol / L paraquat and SBP-3A (low-dose group: 2mg / mL, high-dose group: 4mg / mL). After 5 days of drug administration and culture, the number of dead nematodes was recorded, and the survival rate of nematodes in each group was calculated.

[0118] The experimental results are shown in Table 3 and Figure 7 -A:

[0119] Table 3 Survival rate determination results

[0120] Survival rate (%) Blank control group <![CDATA[100±0 a ]]> Model Group <![CDATA[57.6±5.80 b ]]> Low-dose polysaccharide group <![CDATA[77±5.78 c ]]> High-dose polysaccharide group <![CDATA[87.2±4.40 c ]]>

[0121] As shown in Table 3, paraquat injury significantly reduced the survival rate of nematodes compared with nematodes not treated with polysaccharides. However, nematodes fed with SBP-3A gradually rescued the reduced survival rate in a dose-dependent manner.

[0122] 3.2. Lipofuscin determination results:

[0123] Lipofuscin particles are the final product of lipid peroxidation and usually accumulate during aging and oxidative stress. They are a promising marker of aging. SBP-3A intervention enhanced the clearance of lipofuscin in nematodes. The experimental results are shown in Table 4. Figure 7 -C and Figure 7 -D:

[0124] Table 4 Lipofuscin determination results

[0125] Lipofuscin fluorescence intensity Blank control group <![CDATA[8342.4±1599.13 a ]]> Model Group <![CDATA[14215.8±1284.86 b ]]> Low-dose polysaccharide group <![CDATA[12362.2±1204.82 a ]]> High-dose polysaccharide group <![CDATA[9614±1185.82 a ]]>

[0126] As shown in Table 4, the SBP-3A provided by the present invention gradually eliminated the occurrence of accelerated accumulation of lipofuscin induced by paraquat. Exogenous SBP-3A offsets the oxidative stress triggered by paraquat and endows nematodes with a defense effect against paraquat damage.

[0127] 3.3 ROS measurement results:

[0128] ROS can cause the production of toxic substances such as oxygen free radicals and lipid decomposition products through cellular oxidative stress reactions. The accumulation of ROS in the body can lead to cell death and oxidative damage to the body. In this experiment, DCFH-DA was used to detect ROS. DCFH-DA can freely pass through the cell membrane and is hydrolyzed by esterases in the cell to generate DCFH. However, DCFH cannot pass through the cell membrane and is oxidized to generate the green fluorescent substance DCF in the presence of reactive oxygen. The fluorescence intensity is proportional to the level of reactive oxygen in the cell.

[0129] This experiment detected the accumulation of ROS in nematode cells within 2 h. The experimental results are shown in Table 5 and Figure 7 -B:

[0130] Table 5 ROS determination results

[0131] ROS intensity Blank control group <![CDATA[228.2±6.00 a ]]> Model Group <![CDATA[365.2±4.92 b ]]> Low-dose polysaccharide group <![CDATA[272.7±12.30 c ]]> High-dose polysaccharide group <![CDATA[260.8±3.87 c ]]>

[0132] As shown in Table 5, the ROS content in the polysaccharide group was lower than that in the control group, indicating that SBP-3A can reduce the ROS level in nematodes and enhance the antioxidant capacity of nematodes. Considering that under oxidative stress conditions, SBP-3A intervention has a positive regulatory effect on the production of ROS.

[0133] 3.4. Results of determination of oxidative stress and antioxidant biomarkers:

[0134] The experimental results are shown in Table 6 and Figure 8 As shown:

[0135] Table 6 Results of determination of oxidative stress and antioxidant biomarkers

[0136] CAT activity MDA activity NO activity SOD activity GSH activity Blank control group <![CDATA[6.36±0.13 a ]]> <![CDATA[0.77±0.02 a ]]> <![CDATA[0.77±0.02 a ]]> <![CDATA[31.06±2.95 a ]]> <![CDATA[3.38±0.14 a ]]> Model Group <![CDATA[4.65±0.25 b ]]> <![CDATA[1.09±0.01 b ]]> <![CDATA[1.58±0.10 b ]]> <![CDATA[17.88±1.15 b ]]> <![CDATA[1.97±0.07 c ]]> Low-dose polysaccharide group <![CDATA[5.15±0.14 c ]]> <![CDATA[0.99±0.02 b ]]> <![CDATA[1.28±0.08 b ]]> <![CDATA[25.83±1.15 c ]]> <![CDATA[2.46±0.34 bc ]]> High-dose polysaccharide group <![CDATA[5.59±0.05 d ]]> <![CDATA[0.73±0.04 a ]]> <![CDATA[0.94±0.14 a ]]> <![CDATA[26.43±1.17 c ]]> <![CDATA[2.83±0.13 ab ]]>

[0137] As shown in Table 6, compared with the control group, paraquat treatment significantly reduced the activities of SOD, CAT and GSH. However, compared with the addition of paraquat alone, the exogenous addition of polysaccharides (4 mg / mL) and paraquat significantly increased the activities of antioxidant enzymes. Compared with the model group, the activities of SOD, CAT and GSH in the high-dose polysaccharide group increased by 47.8%, 20.2% and 43.6%, respectively. The results showed that SBP-3A has a protective effect on the antioxidant stress of antioxidant enzymes.

[0138] MDA (malondialdehyde) is the end product of free radicals in the body that cause lipid peroxidation. It is often used to evaluate the body's lipid peroxidation process and the degree of oxidative damage. The higher the MDA content, the more serious the oxidative damage to the body. Figure 8 It can be seen that the MDA content of nematodes in the high-dose polysaccharide group was significantly reduced by 32.8% compared with the model group, indicating that SBP-3A can reduce the degree of lipid peroxidation cell damage in nematodes.

[0139] 3.5. Results of egg laying number determination:

[0140] The total egg production of nematodes and the daily egg production of each group of nematodes were compared to analyze the effect of SBP-3A on the reproductive capacity of nematodes. Fig. 9 As shown, at 20°C, the number of eggs laid by nematodes fed with SBP-3A OP50 was not significantly different from that of the blank control group, indicating that the polysaccharide had no toxic effect on the reproductive ability of nematodes.

Claims

1. A method for preparing beet polysaccharide, characterized in that: The following steps are involved: Step S1. crushing the dried beet pulp to obtain beet pulp powder, uniformly dispersing the beet pulp powder in distilled water to obtain a raw material liquid, adjusting the pH value of the raw material liquid, stirring at 70-90° C. for 1-2 hours, cooling to room temperature, filtering, centrifuging at 6000-10000 g for 18-22 minutes, collecting the supernatant, fully mixing the supernatant with anhydrous ethanol, centrifuging at 6000-10000 g for 20 minutes, collecting the precipitate, washing the precipitate with 95% anhydrous ethanol for 2-4 times, and drying at 40-50° C. for 24 hours to obtain a crude extract of beet pectin; Step S2. dissolving the crude extract of beet pectin obtained in step S1 in distilled water to obtain a crude polysaccharide solution, adding pectinase to the crude polysaccharide solution, adjusting the pH of the crude polysaccharide solution to 3.5, performing enzymolysis at 45-55° C. for 24 hours, then heating in boiling water for 4-6 minutes, centrifuging at a speed of 5000 rpm for 4-5 minutes, collecting the supernatant, then adding trypsin to the supernatant, adjusting the pH of the supernatant to 7, performing enzymolysis at 35-40° C. for 12 hours, inactivating the enzyme, and centrifuging at a speed of 5000 rpm for 5 minutes to obtain a deproteinized solution; Step S3. Apply the deproteinized solution obtained in step S2 to a DEAE-52 agarose column, and gradually elute with distilled water, 0.2 mol / L and 0.4 mol / L sodium chloride, collect the eluate with 0.4 mol / L sodium chloride, further purify on Sephadex G-100, elute with distilled water, detect the polysaccharide content, collect the main peak components, and lyophilize to obtain; In step S1, the pH of the raw material solution is adjusted to 1-2; In step S1, the mixing volume ratio of the supernatant and anhydrous ethanol is 1:3, and the mixture is mixed at 4° C. for 12 h; In the step S2, the amount of pectinase added is 0.2% (m / v) of the volume of the crude polysaccharide solution, and the amount of trypsin added is 0.2% (m / v) of the volume of the crude polysaccharide solution.

2. The method for preparing beet polysaccharide according to claim 1, characterized in that: In step S1, the material-liquid ratio of beet pulp powder to distilled water is 1 g:25 mL.

3. The method for preparing beet polysaccharide according to claim 1, characterized in that: In the step S2, the solid-liquid ratio of the crude beet pectin extract to distilled water is 10 mg:1 mL.

4. The method for preparing beet polysaccharide according to claim 1, characterized in that: The specification of the DEAE-52 agarose column in step S3 is 3.5×40 cm, and the elution rate is 1 mL / min.

5. The method for preparing beet polysaccharide according to claim 1, characterized in that: The specification of Sephadex G-100 in step S3 is 2.4×40 cm, and the elution rate is 0.5 mL / min.

6. Use of the beet polysaccharide prepared by the method for preparing beet polysaccharide according to any one of claims 1 to 5 in the preparation of antioxidant products.

Images