Tartary buckwheat vinasse polysaccharide with antioxidant activity and preparation method thereof

Abstract

The invention discloses tartary buckwheat vinasse polysaccharide with antioxidant activity and a preparation method of the tartary buckwheat vinasse polysaccharide, and belongs to the technical field of polysaccharide. The molecular weight of the tartary buckwheat vinasse polysaccharide with the antioxidant activity is 3.84 * 10 < 5 >, and the monosaccharide is prepared from Man-mannose, Rib-ribose, Rham-rhamnose, GlcUA-glucuronic acid, GalUA-galacturonic acid, Glc-glucose, Gal-galactose, Xyl-xylose and Ara-arabinose according to the molar ratio of (41.60): (20.20): (1.70): (2.84): (4.45): (8.90): (14.39): (6.27): (5.82). The tartary buckwheat vinasse polysaccharide is subjected to crude extraction through an ultrasonic-assisted water extraction and alcohol precipitation method, the extracted crude polysaccharide is purified through ion exchange column and sephadex column chromatography, and the prepared tartary buckwheat vinasse polysaccharide has a good scavenging effect on ABTS < + > free radicals, DPPH free radicals and hydroxyl free radicals and has antioxidant activity.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharides, and particularly relates to tartary buckwheat distiller's grains polysaccharide with antioxidant activity and a preparation method thereof. Background Art

[0002] Buckwheat, as a plant that is both a medicine and an food, has high nutritional and medicinal value and can be used as medicine or food. Buckwheat wine brewed with buckwheat as raw material is a traditional drink of many ethnic minorities, and buckwheat lees are its main brewing by-product.

[0003] Studies have shown that during the distillation process, some non-volatile components in the fermented materials, such as phenols, polysaccharides and other natural active substances, basically remain in the lees. At present, the research on buckwheat lees is mainly focused on the processing of feed and the extraction of flavonoid compounds, while there is little research on the polysaccharides in buckwheat lees. As a natural macromolecular substance with biological activity, polysaccharides have broad development prospects. Buckwheat lees are also rich in highly active polysaccharides. Therefore, studying buckwheat lees polysaccharides can help improve the secondary utilization of lees. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a bitter buckwheat distiller's grains polysaccharide with antioxidant activity and a preparation method thereof

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] In the first aspect, the present invention provides a bitter buckwheat lees polysaccharide with antioxidant activity, the molecular weight of which is 3.84×10 5 The molar ratio of its monosaccharide composition is: Man-mannose: Rib-ribose: Rham-rhamnose: GlcUA-glucuronic acid: GalUA-galacturonic acid: Glc-glucose: Gal-galactose: Xyl-xylose: Ara-arabinose = 41.60: 20.20: 1.70: 2.84: 4.45: 8.90: 14.39: 6.27: 5.82.

[0007] When the concentration of the above-mentioned bitter buckwheat distiller's grains polysaccharide with antioxidant activity was 0.5 mg / mL, the scavenging rates of ABTS+ free radicals, DPPH free radicals and hydroxyl free radicals were 86.47±0.89%, 85.38±2.72% and 90.4±0.02%, respectively.

[0008] In a second aspect, the present invention provides a method for separating and purifying the above-mentioned tartary buckwheat vinasse polysaccharides with antioxidant activity, wherein the tartary buckwheat vinasse polysaccharides are crudely extracted by ultrasonic-assisted water extraction and alcohol precipitation, and then the extracted crude polysaccharides are purified by ion exchange column and dextran gel column chromatography.

[0009] Furthermore, the preparation method of tartary buckwheat distiller's grains polysaccharide with antioxidant activity specifically comprises the following steps:

[0010] S1. dissolving tartary buckwheat lees powder with deionized water and obtaining an extract by ultrasonic extraction;

[0011] S2. The protein in the extract is removed by the Sevage method, and then the water extraction and alcohol precipitation method is used for extraction, the precipitate is collected and re-dissolved with deionized water, heated to remove ethanol and then freeze-dried to obtain crude polysaccharide;

[0012] S3. Prepare a crude polysaccharide solution, load it onto a DEAE-52 cellulose chromatography column, perform gradient elution with NaCl solutions of different concentrations, and detect the absorbance of the eluate at 488 nm using the phenol-sulfuric acid method. There are three elution peaks, which are recorded as TBP-1, TBP-2, and TBP-3;

[0013] S4. After the TBP-2 component is concentrated, it is transferred to a dialysis bag and dialyzed with distilled water;

[0014] S5. Dialysis: The component TBP-2 is chromatographed on a Sephadex G-100 column and eluted with deionized water to obtain a single peak, which is then concentrated and freeze-dried to obtain Tartary buckwheat lees polysaccharide TBP-2 with antioxidant activity.

[0015] In the above step S1, the tartary buckwheat distiller's grains powder is obtained by drying the fermented tartary buckwheat distiller's grains at 50-60° C. to a constant weight, crushing the tartary buckwheat distiller's grains and passing through a 60-mesh sieve.

[0016] In the above step S1, the material-liquid ratio of bitter buckwheat vinasse powder to deionized water is 1:30-40; the temperature of ultrasonic extraction is 50-60° C., the power is 600-700 W, and the time is 55-65 min.

[0017] Furthermore, the solid-liquid ratio of bitter buckwheat distiller's grains powder to deionized water is 1:30; the temperature of ultrasonic extraction is 50°C, the power is 600W, and the time is 60min.

[0018] In the above step S2, the Sevage method is: concentrating the extract to 1 / 2 of the original volume, adding 1 / 2 of the sample volume of Sevage reagent, removing the lower and middle protein layers after centrifugation, and repeating the above operation until no protein layer appears; the volume ratio of dichloromethane and n-butanol in the Sevage reagent is 1:3-4.

[0019] In the above step S2, the water extraction and alcohol precipitation method is: adding anhydrous ethanol to the protein-removed extract, standing at 0-4°C for 20-25 hours to obtain an alcohol precipitation solution, and collecting the precipitate by centrifugation; the volume ratio of the extract to anhydrous ethanol is 1:3-4.

[0020] In the above step S3, the concentration of the crude polysaccharide solution is 9-10 g / L; the DEAE-52 cellulose column filler is pretreated and then filled into the column, the filler amount is 50 g, the chromatography column specification is 1.6×60 cm, and the column volume is 130 mL; the concentrations of the NaCl solution are 0 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.5 mol / L.

[0021] In the above step S4, the component TBP-2 is dialyzed with a 3500Da dialysis bag for 45 to 50 hours, and the distilled water is replaced every 12 hours.

[0022] In the above step S5, 12 g of G-100 dextran gel was loaded into a 1.2×35 cm chromatography column and eluted with 100 to 150 mL of deionized water at a flow rate of 1 to 2 mL / min.

[0023] The beneficial effects of the present invention are as follows: the present invention establishes a complete and feasible technical route for the extraction, separation and purification, structural characteristics and biological activity research of tartary buckwheat vinasse polysaccharides, and provides a new method for the utilization of vinasse after tartary buckwheat wine fermentation. The present invention adopts a water extraction and alcohol precipitation process, which is simple to operate, low in cost, and can be used for large-scale production. The present invention combines an ion exchange chromatography column and a dextran gel chromatography column with a water extraction and alcohol precipitation method to achieve the separation and purification of tartary buckwheat vinasse polysaccharides, and analyzes the structure of the tartary buckwheat vinasse polysaccharides after separation and purification, providing a certain reference for the separation and purification of tartary buckwheat vinasse polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effects of different extraction methods on the extraction content of polysaccharides from tartary buckwheat lees;

[0025] Figure 2 This is the elution curve of buckwheat lees polysaccharide from DEAE-52 ion exchange chromatography column;

[0026] Figure 3 is the elution curve of TBP-2 from the dextran gel chromatography column;

[0027] Figure 4 is the permeabilization gel chromatogram of TBP-2 molecular weight;

[0028] Figure 5 HPLC chromatograms of standard monosaccharides and TBP-2 derivatization;

[0029] Figure 6 This is the infrared spectrum scan of TBP-2;

[0030] Figure 7 is the H NMR spectrum of TBP-2;

[0031] Figure 8is the scanning electron micrograph of TBP-2 (magnification ×300);

[0032] Fig. 9 This is a diagram for the determination of TBP-2 antioxidant activity. DETAILED DESCRIPTION

[0033] The technical solution and effects of the present invention are further illustrated below through practical examples.

[0034] Example 1 Preparation of Tartary Buckwheat Distiller's Grains Polysaccharide Extract

[0035] The fermented tartary buckwheat lees were dried at 55°C to constant weight, crushed and passed through a 60-mesh sieve to obtain tartary buckwheat lees powder. Different solvents were used to dissolve the tartary buckwheat lees powder, and then ultrasonic extraction was performed to determine the effects of different solvents on the extraction rate of tartary buckwheat lees polysaccharides. The specific process is as follows:

[0036] The buckwheat lees powder was divided into four parts, and deionized water, choline chloride: urea: guaiacol (molar ratio of 4:4:1), choline chloride: ethylene glycol (molar ratio of 1:2), and choline chloride: urea (molar ratio of 1:2) were used as solvents, respectively. The mixture was mixed at a solid-liquid ratio of 1:30 (g / mL), and then ultrasonically treated at 50°C with a power of 600 W for 1 h to obtain an extract.

[0037] The polysaccharide content in the polysaccharide extracts of bitter buckwheat lees prepared with different solvents was detected. Figure 1 As shown. The comparison results show that the extraction amount of tartary buckwheat vinasse polysaccharide is the largest when deionized water is used as the extraction solvent. Therefore, the present invention preferably uses deionized water as the solvent to prepare the tartary buckwheat vinasse polysaccharide extract. Compared with deep eutectic melt-based extraction, the method of the present invention is simpler to operate and has no pollution to the environment.

[0038] Example 2 Isolation and purification of bitter buckwheat lees polysaccharides

[0039] S1 concentrated the extract to 1 / 2 of the original volume, added 1 / 2 sample volume of Sevage reagent (n-butanol: dichloromethane volume ratio of 1:4), centrifuged and removed the lower and middle protein layers, repeated three times, added anhydrous ethanol to the protein-removed extract, let stand at 4°C for 24 hours to obtain an alcohol precipitation solution, centrifuged and collected the precipitate, then dissolved the precipitate in deionized water, removed the ethanol at 60°C, and freeze-dried to obtain the initially purified crude polysaccharide of buckwheat lees.

[0040] S2 pretreated 50 g of DEAE-52 cellulose column filler and loaded it into a 1.6×60 cm chromatography column. The crude polysaccharide from buckwheat lees was prepared into a 10 g / L solution with deionized water and then loaded onto the column. Gradient elution was performed with different concentrations of NaCl solution (concentrations were 0 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.5 mol / L). The elution volume was about 130 mL, and the absorbance of the eluate at 488 nm was detected by the phenol-sulfuric acid method.

[0041] The elution curve of the DEAE-52 ion exchange chromatography column of bitter buckwheat distiller's grains polysaccharide is as follows Figure 2 As shown, there are three elution peaks, which are respectively recorded as TBP-1, TBP-2 and TBP-3.

[0042] S3 dialyze component TBP-2 using a 3500Da dialysis bag for 48 hours, replacing distilled water every 12 hours.

[0043] S4: Load 12 g of Sephadex G-100 dextran gel into a 1.2×35 cm chromatography column and elute with 100 mL of deionized water at a flow rate of 2 mL / min. Further purify the component TBP-2, concentrate and freeze-dry to obtain purified TBP-2.

[0044] The elution curve of TBP-2 polysaccharide from bitter buckwheat grains on the dextran gel chromatography column is as follows: Figure 3 As shown, a single peak was obtained, which can be used for subsequent structure determination and activity experiments.

[0045] Example 3 Physical and chemical properties test of bitter buckwheat lees polysaccharide TBP-2

[0046] The buckwheat vinasse polysaccharide TBP-2 was prepared in the above Example 1. In order to analyze the structure and antioxidant activity of the buckwheat vinasse polysaccharide TBP-2, the following experiment was conducted.

[0047] 1. Molecular weight determination

[0048] Gel permeation chromatography was connected to a differential refractive index detector. The sample was filtered through a 0.22 μm filter membrane before injection. The molecular weight of TBP-2 polysaccharide from bitter buckwheat grains was analyzed by gel permeation chromatography. Figure 4 As shown in Figure 2, the molecular weight of TBP-2 from bitter buckwheat lees polysaccharide was 3.84×10 5 .

[0049] 2. Monosaccharide composition of TBP-2

[0050] 100 μL of 4 g / L buckwheat lees polysaccharide TBP-2 was taken, 100 μL of 4 g / L trifluoroacetic acid (TFA) was added, the tube was sealed with N2, hydrolyzed at 110°C for 4 h, 200 μL of methanol was added after cooling, and the mixture was dried with N2 to remove TFA, 50 μL of sodium hydroxide solution was added to dissolve the solid, and 0.5 mol / L PMP methanol solution was added, and the reaction was carried out at 700°C for 100 min to complete the derivatization.

[0051] The monosaccharide composition was determined by high performance liquid chromatography, with mobile phase: C was 0.1 mol / L phosphate buffer (pH 6.7), D was acetonitrile, isocratic elution (83:17), column temperature was 30°C, detection wavelength was 250 nm, and flow rate was 1 mL / min. Figure 5 As shown, Man-mannose, Rib-ribose, Rham-rhamnose, GlcUA-glucuronic acid, GalUA-galacturonic acid, Glc-glucose, Gal-galactose, Xyl-xylose, Ara-arabinose are composed. According to the peak area, the corresponding molar percentage can be obtained: 41.60:20.20:1.70:2.84:4.45:8.90:14.39:6.27:5.82. Among them, mannose, ribose, D-galactose, xylose and arabinose are the main components.

[0052] 3.TBP-2 infrared spectrum scanning

[0053] 5 mg polysaccharide sample was mixed with potassium bromide and pressed into tablets. The results were analyzed by BRUKER infrared spectrometer at 4500-800 cm -1 scanning.

[0054] Figure 6 This is the infrared spectrum scan of TBP-2, 3405cm -1 The strong absorption peak near 2922cm indicates the presence of OH stretching vibration; -1 The absorption peak at 1637cm is the stretching vibration of CH, which may contain -CH3 or -CH4 groups; -1 1416cm-1 is the stretching vibration peak of the carboxylate COO carbonyl (C=O), which confirms the existence of TBP-2 galacturonic acid, and it may exist mainly in the form of COO-; -1 is the in-plane bending vibration peak of CH; 1340cm -1 The absorption peak appears at 845cm -1 is the absorption peak of α-glycosidic bond, 917.64 cm -1 is the absorption peak of β-glycosidic bond, 762cm -1It is the characteristic absorption peak of the pyranose ring, which indicates that TBP-2 is a pyranose containing α-glycosidic bonds and β-glycosidic bonds, and also proves the presence of mannose.

[0055] 4. TBP-2 NMR

[0056] Figure 7 This is the nuclear magnetic resonance hydrogen spectrum of the purified bitter buckwheat lees polysaccharide TBP-2. Generally, the chemical shift of the α-glycosidic bond is greater than 5.0 ppm, and the chemical shift of the β-anomeric hydrogen is less than 5.0 ppm. Figure 7 It can be seen that the anomeric hydrogen signal of TBP-2 is in the δ3.0ppm-5.5ppm region, which is a typical feature of polysaccharides. Among them, the shifted proton signals were observed near 5.1 and 4.4ppm, indicating that TBP-2 is a pyranose ring polysaccharide connected by α-glycosidic bonds and β-glycosidic bonds. There are strong signals in the range of 3.20-3.50ppm, which is related to the presence of more -OCH3 in the polysaccharide, and also indicates that it contains galactose H2-H6. 5.1 and 3.43ppm indicate that the polysaccharide has H1 and H2 proton signals of α-D-glucose residues. At the same time, the proton signal at 5.5ppm is the proton signal of arabinose, and the strong signal at 3.9ppm is the anomeric proton signal of galacturonic acid, which is consistent with the results of infrared spectroscopy detection.

[0057] 5.TBP-2 Scanning Electron Microscope

[0058] Figure 8 The microscopic state of Tartary Buckwheat lees polysaccharide before and after purification under 300 times magnification. The surface of crude Tartary Buckwheat lees polysaccharide fragments before purification (left) is slightly uneven and has holes; after purification (right), Tartary Buckwheat lees polysaccharide TBP-2 is granular and presents an accumulation state. Compared with crude polysaccharide, the density and smoothness of Tartary Buckwheat lees TBP-2 polysaccharide after purification are significantly improved.

[0059] 6. Antioxidant activity of TBP-2 polysaccharide from bitter buckwheat grains

[0060] 10mg, 20mg, 30mg, 40mg, and 50mg of TBP-2 were selected and dissolved in 100mL ultrapure water to obtain sample solutions of different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5mg / mL). The corresponding concentration of Vc was used as a positive control to determine the scavenging rate of hydroxyl radicals, ABTS+, and DPPH free radicals and the iron ion reducing power of TBP-2.

[0061] Fig. 9This is the antioxidant activity determination diagram of TBP-2. The antioxidant capacity of the separated and purified bitter buckwheat lees polysaccharide TBP-2 increases with the increase of mass concentration. Compared with Vc, it has a better scavenging effect on ABTS+ free radicals, DPPH free radicals and hydroxyl free radicals, with scavenging rates of 86.47±0.89%, 85.38±2.72% and 90.4±0.02% respectively; while the reduction effect on iron ions is poor. This shows that the separated and purified bitter buckwheat lees polysaccharide has a certain antioxidant effect. It can be considered to be added to the wine body in the future to make a wine with health benefits, thereby improving the utilization value of bitter buckwheat lees.

Claims

1. Tartary buckwheat lees polysaccharide with antioxidant activity, characterized by: The molecular weight is 3.84×10 5 ; The molar ratio of its monosaccharide composition is: Man-mannose: Rib-ribose: Rham-rhamnose: GlcUA-glucuronic acid: GalUA-galacturonic acid: Glc-glucose: Gal-galactose: Xyl-xylose: Ara-arabinose = 41.60: 20.20: 1.70: 2.84: 4.45: 8.90: 14.39: 6.27: 5.

82.

2. The tartary buckwheat lees polysaccharide with antioxidant activity according to claim 1, characterized in that: When the concentration of the bitter buckwheat lees polysaccharide TBP-2 is 0.5 mg / mL, the scavenging rates of the ABTS+ free radical, DPPH free radical and hydroxyl free radical are 86.47±0.89%, 85.38±2.72% and 90.4±0.02% respectively.

3. A method for preparing bitter buckwheat lees polysaccharide with antioxidant activity, characterized in that: The steps include: S1. dissolving buckwheat lees powder with deionized water and obtaining an extract by ultrasonic extraction; S2. The protein in the extract is removed by the Sevage method, and then the water extraction and alcohol precipitation method is used for extraction, the precipitate is collected and re-dissolved with deionized water, heated to remove ethanol and then freeze-dried to obtain crude polysaccharide; S3. Prepare a crude polysaccharide solution, load it onto a DEAE-52 cellulose chromatography column, perform gradient elution with NaCl solutions of different concentrations, and detect the absorbance of the eluate at 488 nm using the phenol-sulfuric acid method. There are three elution peaks, which are recorded as TBP-1, TBP-2, and TBP-3; S4. After the TBP-2 component is concentrated, it is transferred to a dialysis bag and dialyzed with distilled water; S5. Dialysis: The component TBP-2 is chromatographed on a Sephadex G-100 column and eluted with deionized water to obtain a single peak, which is then concentrated and freeze-dried to obtain buckwheat lees polysaccharide with antioxidant activity.

4. The method for preparing tartary buckwheat lees polysaccharide with antioxidant activity according to claim 3, characterized in that: In step S1, the tartary buckwheat distiller's grains powder is obtained by drying the fermented tartary buckwheat distiller's grains at 50-60° C. to a constant weight, crushing the tartary buckwheat distiller's grains and passing through a 60-mesh sieve.

5. The method for preparing tartary buckwheat lees polysaccharide with antioxidant activity according to claim 3, characterized in that: In step S1, the material-liquid ratio of bitter buckwheat lees powder to deionized water is 1:30-40; the temperature of ultrasonic extraction is 50-60° C., the power is 600-700 W, and the time is 55-65 min; Preferably, the solid-liquid ratio of bitter buckwheat vinasse powder to deionized water is 1:30; the temperature of ultrasonic extraction is 50° C., the power is 600 W, and the time is 60 min.

6. The method for preparing tartary buckwheat lees polysaccharide with antioxidant activity according to claim 3, characterized in that: In step S2, the Sevage method is as follows: the extract is concentrated to 1 / 2 of the original volume, 1 / 2 of the sample volume of Sevage reagent is added, the lower and middle protein layers are removed after centrifugation, and the above operation is repeated until no protein layer appears; the volume ratio of dichloromethane to n-butanol in the Sevage reagent is 1:3-4.

7. The method for preparing tartary buckwheat lees polysaccharide with antioxidant activity according to claim 3, characterized in that: In step S2, the water extraction and alcohol precipitation method is as follows: anhydrous ethanol is added to the protein-removed extract, and the solution is allowed to stand at 0-4°C for 20-25 hours to obtain an alcohol precipitation solution, and the precipitate is collected by centrifugation; the volume ratio of the extract to anhydrous ethanol is 1:3-4.

8. The method for preparing tartary buckwheat lees polysaccharide with antioxidant activity according to claim 3, characterized in that: In step S3, the concentration of the crude polysaccharide solution is 9-10 g / L; the DEAE-52 cellulose column filler is pretreated and then filled into the column, the filler amount is 50 g, the chromatography column specification is 1.6×60 cm, and the column volume is 130 mL; the concentrations of the NaCl solution are 0 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.5 mol / L.

9. The method for preparing tartary buckwheat lees polysaccharide with antioxidant activity according to claim 3, characterized in that: In step S4, the component TBP-2 is dialyzed using a 3500Da dialysis bag for 45 to 50 hours, and the distilled water is replaced every 12 hours.

10. The method for preparing tartary buckwheat lees polysaccharide with antioxidant activity according to claim 3, characterized in that: In step S5, 12 g of G-100 polysaccharide gel is loaded into a 1.2×35 cm chromatography column and eluted with 100 to 150 mL of deionized water at a flow rate of 1 to 2 mL / min.

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