Extraction method and application of pot-bottom water polysaccharide and combined choline chloride-fructose deep eutectic solvent of pot-bottom water polysaccharide
Through the deep eutectic solvent of choline chloride-fructose and multi-step separation technology, the polysaccharides in the water of the liquor brewing base pot were successfully extracted and identified, solving the problems of cumbersome extraction process and unstable structure in the existing technology, and realizing the polysaccharides Efficient purification and antioxidant activity analysis have laid a theoretical foundation for its application in functional products.
Patent Information
- Application Number
- CN202411451707.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-17
- Publication Date
- 2024-12-17
AI Technical Summary
In the prior art, the polysaccharide extraction process in the base pot of liquor brewing is complicated and costly, and it is difficult to ensure that the structure and activity of the polysaccharides are not damaged. There is a lack of research on the separation and identification of polysaccharides in the base pot.
Liquid-liquid extraction was performed using choline chloride-fructose deep eutectic solvent, combined with standstill, centrifugation, alcohol precipitation, dialysis, anion exchange column and dextran gel column chromatography, and separating polysaccharides from the bottom pot. , and its structural characteristics and functional activities are identified through scanning electron microscopy, infrared and nuclear magnetic technologies.
It realizes efficient extraction and purification of polysaccharides in water in the pot, ensures the structural integrity of the polysaccharides, provides a basis for its antioxidant activity analysis, provides theoretical support for the application of food, health care drugs and cosmetics, and makes up for the separation and identification in the existing technology. blank.
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Figure CN119143897A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of active substance extraction, and in particular relates to a bottom pot water polysaccharide and an extraction method and application thereof using a combined choline chloride-fructose deep eutectic solvent. Background Art
[0002] Baijiu (Chinese liquor) is a distilled liquor made from raw materials such as sorghum, rice, wheat, peas, and corn. It is produced using fermentation agents through a series of processes, including raw material crushing, fermentation, distillation, storage, and blending. Baijiu (Chinese liquor) has a long history and is a large-scale production industry in my country. In recent years, with the rapid development of the baijiu industry, the production of byproducts (such as distiller's grains and wastewater) has also increased significantly. Distillery wastewater is a major byproduct resource in the baijiu production process. According to incomplete statistics, approximately 48 tons of wastewater are generated for every ton of baijiu produced. Bottom-pot water is a major source of this wastewater. This is formed when water vapor repeatedly condenses in the grain mash during grain gelatinization and mash distillation, sinks, accumulates, and mixes with the existing water at the bottom of the pot. Furthermore, during actual production, yellow water is added to the bottom pot during distillation to increase liquor yield. This bottom-pot water is typically yellowish-brown or brown. The composition of bottom pot water is complex and diverse, containing organic substances such as starch, reducing sugars, alcohols, and organic acids. If it is directly treated, it will not only be difficult and costly, but will also lead to the organic components not being fully utilized, resulting in waste of resources. Therefore, the resource utilization of bottom pot water is of great significance.
[0003] With the rise of the concept of healthy eating, the extraction of natural active ingredients for use in functional foods and medicines has received widespread attention. Polysaccharides are a class of natural macromolecular polymers composed of many identical or different monosaccharides connected by α / β-glycosidic bonds. They have multiple activities such as antioxidant, anti-inflammatory and hypoglycemic, and are widely used in clinical treatment and prevention of diseases.
[0004] There are few existing technologies for the resource utilization of baijiu brewing pan water. For example, invention CN115160030A uses pan water to produce liquid organic fertilizer containing γ-polyglutamic acid; invention CN114315046A uses brewing wastewater as raw material to prepare bioactive fermentation filtrate, thereby effectively utilizing the effective components therein; invention CN116769840A discloses a method for producing Chlorella growth factor using pan water. As for the preparation of active components from pan water, only invention CN1176999876A discloses a method for resource utilization of pan water from brewing, in which a crude polysaccharide extraction method is established. Although this method recovers the beneficial components of polysaccharides in pan water to a certain extent, the following problems still exist: (1) The extraction process is cumbersome, and industrial application and production are difficult and costly; (2) The extraction process requires concentration. If the concentration temperature cannot be well controlled, it is difficult to ensure that the polysaccharide structure and activity are not destroyed; (3) This technology is limited to the preparation of crude extracts of pan water polysaccharides, and its separation and identification have not yet been studied.
[0005] Therefore, it is of great practical significance to use green and efficient deep eutectic solvents to separate and purify polysaccharides from water chestnuts, analyze their structural characteristics, explore their functional activities, and provide a theoretical basis for their application in food, health care drugs and cosmetics. Summary of the Invention
[0006] The present invention aims to overcome the shortcomings of the existing technology and provide a method for extracting and isolating polysaccharides from radix serrata using a natural deep eutectic solvent (NaDES). This method utilizes NaDES extraction, alcohol precipitation, dialysis, ion exchange column chromatography, and dextran gel column chromatography to separate and purify radix serrata polysaccharides. The polysaccharide structure is identified using scanning electron microscopy, infrared spectroscopy, and nuclear magnetic resonance (NMR) techniques, while also exploring its in vitro antioxidant activity.
[0007] To achieve the above objectives, the present invention first provides a method for extracting polysaccharides from sorghum sorghum using a combined choline chloride-fructose deep eutectic solvent, which comprises the following steps:
[0008] A. After centrifuging the brewing bottom pot water, the centrifuged bottom pot water is mixed with a deep eutectic solvent, and liquid-liquid extraction is performed under ultrasonic conditions to obtain an extract. In step A, the deep eutectic solvent is prepared by the following method: choline chloride and fructose are mixed uniformly in a molar ratio of 1:1 to 4, water is added in an amount of 10 to 40% of the total mass of choline chloride and fructose, and heating and stirring (to promote the formation of hydrogen bonds between the components) until a uniform, stable, clear and transparent liquid is formed, thereby obtaining choline chloride-fructose NaDES.
[0009] B. Cooling, alcohol precipitation, solid-liquid separation and freeze-drying the extract obtained in step A to obtain crude polysaccharide;
[0010] C. After the crude polysaccharide of the bottom pot water obtained in step B is prepared into an aqueous solution, Sevage reagent is added to remove protein, the upper sugar solution is collected, and then soluble small molecules are removed by dialysis. The dialyzed solution is freeze-dried to obtain the bottom pot water polysaccharide DGSP.
[0011] Among them, in the above-mentioned extraction method, in step A, the specific operation of standing and filtering the brewing bottom pot water is: after the brewing bottom pot water is stood at 0-4°C for 24-48 hours, it is centrifuged at least once, and the first centrifugation conditions are: temperature 0-4°C, speed 8000-10000rpm, time 15-20min; subsequent centrifugation conditions: temperature 0-4°C, speed 5000-8000rpm, time 10-15min.
[0012] Wherein, in the above extraction method, in step A, the volume ratio of the bottom pot water after centrifugation to the deep eutectic solvent is 1:1-2.
[0013] Wherein, in the above extraction method, in step A, the power of the ultrasound is 200-400W.
[0014] Wherein, in the above extraction method, in step A, the temperature of the liquid-liquid extraction is 40-60°C.
[0015] Wherein, in the above extraction method, in step A, the liquid-liquid extraction time is 40 to 60 minutes.
[0016] Preferably, in the above extraction method, in step A, when preparing the deep eutectic solvent, the molar ratio of choline chloride to fructose is 1:1-2.
[0017] Preferably, in the above extraction method, in step A, when preparing the deep eutectic solvent, water is added in an amount of 10 to 20% of the total mass of choline chloride and fructose.
[0018] Wherein, in the above extraction method, in step A, when preparing the deep eutectic solvent, the heating and stirring temperature is 80-90°C.
[0019] Wherein, in the above extraction method, in step A, when preparing the deep eutectic solvent, the heating and stirring time is 5 to 6 hours.
[0020] Wherein, in the above extraction method, in step B, the alcohol precipitation is: mixing the extract and anhydrous ethanol in a volume ratio of 1:3-5, and letting it stand at 0-4°C for 24-48 hours.
[0021] Wherein, in the above extraction method, in step B, the solid-liquid separation is carried out by centrifugation.
[0022] Preferably, in the above extraction method, in step B, during solid-liquid separation, the centrifugation is: centrifuging at a temperature of 0 to 4° C. and a rotation speed of 8000 to 10000 rpm for 15 to 20 minutes to collect the precipitate.
[0023] Wherein, in the above extraction method, in step B, the freeze-drying conditions are: temperature -80 to -60°C, vacuum degree 1 to 2 Pa, and time 24 to 48 hours.
[0024] In the above extraction method, in step C, the crude polysaccharide from the bottom pot water is prepared into an aqueous solution with a concentration of 10 to 20 mg / mL.
[0025] In the above extraction method, in step C, the volume ratio of the crude polysaccharide aqueous solution in bottom pot water to the Sevage reagent is 4 to 5:1.
[0026] Wherein, in the above extraction method, in step C, the upper sugar solution is collected by centrifugation.
[0027] Preferably, in the above extraction method, when collecting the upper sugar solution, the centrifugation is: centrifuging at a temperature of 0-4°C and a rotation speed of 5000-8000 rpm for 10-15 minutes to collect the upper sugar solution.
[0028] In the above extraction method, in step C, the dialysis is: using a dialysis bag to dialyze at 0-4°C for 36-48 hours to retain components with a molecular weight greater than 3500Da, during which the water is replaced every 6-8 hours.
[0029] Wherein, in the above extraction method, in step C, the freeze-drying conditions are: temperature -80 to -60°C, vacuum degree 1 to 2 Pa, and time 24 to 48 hours.
[0030] The present invention also provides a method for extracting bottom pot water polysaccharides using a combined choline chloride-fructose deep eutectic solvent, which, based on the above method, further comprises the following steps:
[0031] D. After the bottom pot water polysaccharide DGSP obtained in step C is prepared into an aqueous solution, it is first purified by anion exchange column chromatography to obtain bottom pot water polysaccharide DGSPx, and then purified by dextran gel column chromatography to obtain bottom pot water polysaccharide DGSPb-1.
[0032] In the above extraction method, in step D, the bottom pot water polysaccharide DGSP obtained in step C is prepared into an aqueous solution with a concentration of 10 to 20 mg / mL.
[0033] In the above-mentioned extraction method, in step D, the anion exchange column chromatography purification operation is as follows: the bottom pot water polysaccharide DGSP aqueous solution is loaded onto a DEAE52 anion exchange cellulose column, and the eluent is 0-0.4 mol / L NaCl solution in sequence. During this period, the polysaccharide content is determined by the phenol-sulfuric acid method, and the polysaccharide content is determined until no polysaccharide liquid flows out, and the effective polysaccharide components at 0 mol / L, 0.1 mol / L and 0.4 mol / L are collected respectively, and after dialysis and freeze-drying, the bottom pot water polysaccharide DGSPx is obtained. The bottom pot water polysaccharide DGSP includes DGSPa, DGSPb and / or DGSPc, wherein the effective polysaccharide component at 0 mol / L corresponds to DGSPa, the effective polysaccharide component at 0.1 mol / L corresponds to DGSPb, and the effective polysaccharide component at 0.4 mol / L corresponds to DGSPc.
[0034] In step D, during purification by anion exchange column chromatography, DGSPb is the main elution peak, while the other two have lower absorbance values. Therefore, preferably, in the above-mentioned extraction method, in step D, the anion exchange column chromatography purification operation is as follows: the bottom pot water polysaccharide DGSP aqueous solution is loaded onto a DEAE52 anion exchange cellulose column, and the eluent is sequentially 0-0.1 mol / L NaCl solution. During this period, the polysaccharide content is determined by the phenol-sulfuric acid method. The column is continued until no more polysaccharide flows out, and the effective polysaccharide fraction at 0.1 mol / L is collected, dialyzed, and freeze-dried to obtain DGSPb.
[0035] Preferably, in the above extraction method, in step D, during the anion exchange column chromatography purification, the eluent flow rate is 1-2 mL / min, and every 4-5 mL is collected into one tube.
[0036] Preferably, in the above extraction method, in step D, during the anion exchange column chromatography purification, the dialysis is: using a dialysis bag to dialyze for 36 to 48 hours at 0 to 4°C to retain components with a molecular weight greater than 3500Da, during which the water is replaced every 6 to 8 hours.
[0037] Preferably, in the above extraction method, in step D, during the anion exchange column chromatography purification, the freeze-drying conditions are: temperature -80 to -60°C, vacuum degree 1 to 2 Pa, and time 24 to 48 hours.
[0038] In the above extraction method, in step D, the DGSPb obtained by purification by anion exchange column chromatography is prepared into an aqueous solution with a concentration of 10 to 20 mg / mL.
[0039] Among them, in the above-mentioned extraction method, in step D, the operation of the dextran gel column chromatography purification is: the DGSPb obtained by anion exchange column chromatography purification is configured into an aqueous solution, loaded onto Sephadex G-100 dextran gel column chromatography, and the eluent is water. During this period, the polysaccharide content is determined by the phenol-sulfuric acid method. When no polysaccharide liquid flows out, the effective polysaccharide component is collected, dialyzed and freeze-dried to obtain the bottom pot water polysaccharide DGSPb-1.
[0040] Preferably, in the above extraction method, in step D, during the purification by dextran gel column chromatography, the eluent flow rate is 1-2 mL / min, and every 4-5 mL is collected into one tube.
[0041] Preferably, in the above extraction method, in step D, during the dextran gel column chromatography purification, the dialysis is: using a dialysis bag to dialyze for 36 to 48 hours at 0 to 4°C to retain components with a molecular weight greater than 3500Da, during which the water is replaced every 6 to 8 hours.
[0042] Preferably, in the above extraction method, in step D, during the purification by dextran gel column chromatography, the freeze-drying conditions are: temperature -80 to -60°C, vacuum degree 1 to 2 Pa, and time 24 to 48 hours.
[0043] The present invention uses gel permeation chromatography (GPC) to determine the molecular weight of the purified polysaccharide components; scanning electron microscopy, infrared spectroscopy, and nuclear magnetic resonance analysis of the polysaccharide's physical and chemical properties. In the above extraction method, in step D, the molecular weight of the bottom pot water polysaccharide DGSPb-1 is 11.8832 kDa, and the molar ratio of its monosaccharide composition is Glc:Rib:Rha:Man:GlcA:Gal:Xyl:Ara = 26.97:3.03:0.68:52.44:5.31:3.44:2.68:5.45.
[0044] The present invention also provides bottom pot water polysaccharide DGSP, bottom pot water polysaccharide DGSPx and / or bottom pot water polysaccharide DGSPb-1, which are obtained by extraction using the above method.
[0045] The present invention evaluates the antioxidant activity of scalloped water polysaccharides using scavenging 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals and hydroxyl (OH) free radicals, as well as total antioxidant capacity, as indicators. Tests have shown that the scalloped water polysaccharides DGSP, DGSPx, and / or DGSPb-1 of the present invention have excellent free radical scavenging capabilities. Therefore, the present invention also provides the use of the above-mentioned scalloped water polysaccharides DGSP, DGSPx, and / or DGSPb-1 in the preparation of free radical scavengers.
[0046] Preferably, in the above use, the free radical is a DPPH free radical and / or an OH free radical.
[0047] In the present invention, the bottom boiler water of liquor brewing wastewater is a well-known substance in the art, which can be referred to literature 1 (Luo Yuting, Zhang Yuliang, Shui Liangyang, etc. Research on the treatment of bottom boiler water of liquor brewing wastewater with quadriceps algae [J]. Modern Chemical Industry, 2024, 44(02): 206-210), literature 2 (Jiang Handa, Yan Luming, Li Rong, etc. Resource utilization of bottom boiler water of liquor brewing to produce Chlorella growth factor [J]. Chemistry and Bioengineering, 2024, 41(07): 37-43) and other literatures.
[0048] Beneficial effects of the present invention:
[0049] The choline chloride-fructose NaDES solvent prepared by the present invention is green and efficient, and can be used as an excellent extractant for polysaccharides in bottom pot water. Considering the complex composition of bottom pot water, the bottom pot water is first pretreated before extraction. Insoluble impurities in the bottom pot water are removed to the greatest extent possible through methods such as low-temperature stasis and centrifugation. This method is gentle and does not destroy the chemical structure of the active ingredients in the bottom pot water. During the extraction process, bottom pot water and choline chloride-fructose NaDES solvent are added in different volume ratios to adjust the polarity of the extract, ensuring the dissolution of polysaccharide molecules of different polarities in the bottom pot water, thereby improving the extraction content and efficiency.
[0050] In the Sevage reagent, non-toxic dichloromethane is used instead of chloroform, a hazardous chemical, to ensure that no hazardous chemicals are used in the extraction process. At the same time, it can effectively remove impurities such as proteins and pigments in crude polysaccharides, facilitating subsequent analysis. DEAE52 anion exchange cellulose column and Sephadex G-100 dextran gel column chromatography were used to purify the crude polysaccharides from the bottom pot water, and the structure of the purified bottom pot water polysaccharides was determined. The present invention not only effectively extracts polysaccharide substances from the bottom pot water, but also further separates the bottom pot water polysaccharides and analyzes their molecular weight, monosaccharide composition, structure and antioxidant capacity, etc., indicating that the separation and identification method is effective and feasible, and the analysis structure is accurate and reliable, which fills the gap in the existing separation and identification of bottom pot water polysaccharides and solves the problem of separation and identification of bottom pot water polysaccharides in the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS
[0051] Figure 1 This is the DEAE52 anion exchange cellulose column chromatography elution diagram of the bottom pot water polysaccharide DGSP in Example 2.
[0052] Figure 2 This is the elution diagram of Sephadex G-100 dextran gel column chromatography of the bottom pot water polysaccharide DGSPb in Example 2.
[0053] Figure 3 This is the relative molecular mass diagram of the bottom pot water polysaccharide DGSPb-1.
[0054] Figure 4 This is the monosaccharide composition analysis diagram of the bottom pot water polysaccharide DGSPb-1.
[0055] Figure 5 This is the infrared spectrum of the bottom pot water polysaccharide DGSPb-1.
[0056] Figure 6 For the bottom pot water polysaccharide DGSPb-1 1 H NMR spectrum.
[0057] Figure 7These are the DPPH free radical scavenging curve and total antioxidant capacity diagram of the bottom pot water polysaccharide DGSPb-1; among them, A is the DPPH free radical scavenging capacity determination, and B is the total antioxidant capacity determination. DETAILED DESCRIPTION
[0058] The present invention is further described in detail below through examples, but the protection scope of the present invention is not limited to the scope of the examples.
[0059] Example 1
[0060] This embodiment provides a method for preparing crude polysaccharide DGSP from distilled water, and the specific steps are as follows:
[0061] Pretreatment of brewing pot water: After the brewing pot water has been allowed to stand at 0-4°C for 48 hours, take 1L of pot water sample and centrifuge it at 4°C and 10,000rpm for 20 minutes. After collecting the supernatant, continue centrifuging it at 4°C and 8,000rpm for 10 minutes to obtain the centrifuged pot water. The centrifuged pot water can be filtered with ordinary medium-speed filter paper and 0.45μm water filter membrane in turn, and can be stored at 4°C for a long time.
[0062] Preparation of choline chloride-fructose deep eutectic solvent NaDES solvent: choline chloride and fructose are added into a reactor at a molar ratio of 1:2, and then ultrapure water with a total mass ratio of 20% of choline chloride and fructose is added. The mixture is placed in a magnetic stirrer and heated at 80°C for 5 hours to form a uniform, clear and transparent liquid, thereby obtaining choline chloride-fructose NaDES.
[0063] The centrifuged brewing bottom pot water and choline chloride-fructose NaDES solvent were mixed evenly in a volume ratio of 1:2, ultrasonicated at 40°C and 200W for 40 minutes, cooled to room temperature, and then added with 4 times anhydrous ethanol (the final ethanol concentration of the sample was 80%) and allowed to stand at 4°C for 24 hours. After removing 2 / 3 of the supernatant, the mixture was centrifuged (4°C, 10,000 rpm, 15 minutes) to collect the precipitate. The precipitate was placed in a 40°C water bath to remove excess ethanol, and then freeze-dried at -80°C and a vacuum degree of about 1Pa for 48 hours to obtain crude bottom pot water polysaccharide.
[0064] The lyophilized crude polysaccharide from the bottom pot water was dissolved in water to a concentration of 20 mg / mL. Then, 1 / 5 of the volume of the crude polysaccharide solution of the bottom pot water was added with Sevage reagent (Sevage reagent is a mixture of dichloromethane and n-butanol in a volume ratio of 4:1). After mixing evenly, centrifugation was performed (4°C, 8000 rpm, 10 min). The upper sugar solution was collected and repeated several times until no protein appeared in the middle. The bottom pot water polysaccharide sample was then placed in a dialysis bag and dialyzed at 4°C for 48 hours to retain components with a molecular weight greater than 3500 Da. During this period, ultrapure water was replaced every 6 hours. After dialysis, the crude polysaccharide was freeze-dried for 48 hours at -80°C and a vacuum of about 1 Pa to obtain the crude polysaccharide from the bottom pot water, which was recorded as DGSP.
[0065] Example 2
[0066] This example is the separation and purification of polysaccharides from the bottom pot water
[0067] (1) Take the crude polysaccharide DGSP sample obtained in Example 1, dissolve it in ultrapure water, and prepare a 10 mg / mL DGSP solution. Purify it through a DEAE52 anion exchange cellulose column. The eluent is 0, 0.1, 0.2, and 0.4 mol / L NaCl solution in sequence. The flow rate is 2 mL / min. One tube is collected every 6 mL. During this period, the polysaccharide content is determined by the phenol-sulfuric acid method. The elution curve is drawn according to the OD value, and the effective polysaccharide components are collected. The results are as follows. Figure 1 After purification on a DEAE52 anion exchange cellulose column, the polysaccharide solution from the bottom pot water was placed into dialysis bags according to the elution fractions. The solution was dialyzed at 4°C for 48 hours to retain fractions with a molecular weight greater than 3500 Da, with ultrapure water replaced every 6 hours. The dialyzed polysaccharide fractions were then freeze-dried in a vacuum (-80°C, 1 Pa, 48 hours) to obtain three purified polysaccharide fractions, designated DGSPa, DGSPb, and DGSPc.
[0068] (2) The DGSPb obtained in step (1) was prepared into a 10 mg / mL aqueous solution and then subjected to Sephadex G-100 dextran gel column chromatography. The column size was 1.0 cm*40 cm, the eluent was ultrapure water, the flow rate was 1 mL / min, and 4 mL was collected in one tube. During the process, the polysaccharide content was determined by the phenol-sulfuric acid method, and the elution curve was drawn according to the OD value. The results are shown in Figure 2. Figure 2 The effective polysaccharide fractions were collected based on the elution peak and placed in a dialysis bag. They were dialyzed at 4°C for 48 hours to retain fractions with a molecular weight greater than 3500 Da, with ultrapure water replaced every 6 hours. The dialyzed polysaccharide fractions were freeze-dried in a vacuum (-80°C, 1 Pa, 48 hours) to obtain a purified polysaccharide fraction designated DGSPb-1.
[0069] Example 3
[0070] This example is the characterization of the bottom pot water polysaccharide DGSPb-1.
[0071] Molecular weight determination: High performance gel permeation chromatography (HP-GPC) was used to determine the molecular weight of the sample. Chromatographic conditions: Mobile phase: ultrapure water; Flow rate: 0.6 mL / min; Column temperature: 35°C; Injection volume: 20 μL. Dextran with different molecular weights (300,000, 200,000, 150,000, 70,000, 10,000, 3,000, 1,000, 800, 350, 180 Da) was used as a standard. The results are shown in Figure 2. Figure 3 As shown, according to the standard curve and the calculated molecular weight of the sample, the molecular weight of DGSPb-1 was obtained to be approximately 11.8832 kDa (Rt=4.9312).
[0072] Sugar composition determination: Monosaccharide composition was determined using pre-column derivatization HPLC. Hydrolysis: 10 mg of the DGSPb-1 sample obtained in Example 2 was added with 4 mL of 2 mol / L trifluoroacetic acid (TFA). The mixture was immediately sealed with nitrogen and hydrolyzed at 110°C for 4 h. TFA was then removed by nitrogen purge, followed by washing with 5 mL of methanol and drying with nitrogen purge. This process was repeated five times to remove all TFA. The volume was then brought to 5 mL with ultrapure water for later use.
[0073] Derivatization: Take 500 μL of the hydrolyzed sample solution, add 200 μL of 0.3 mol / L NaOH solution and 1 mL of 0.5 mol / L PMP methanol solution, mix well, and react in a 70°C water bath for 100 min. After cooling to room temperature, add 200 μL of 0.3 mol / L HCl and 600 μL of ultrapure water, mix well, and then extract with 1 mL of chloroform. Repeat this process three times. The aqueous phase solution is diluted to 3 mL, filtered through a 0.22 μm microporous filter, and transferred to a chromatographic vial for analysis.
[0074] The monosaccharide composition and content of DGSPb-1 were determined using an Agilent ZORBAX Eclipse Plus C18 column (Analytical 4.6 mm × 250 mm, 5-Micron); the elution conditions were 82% A (0.05 mol / L phosphate buffer solution, pH = 6.8) and 18% B (acetonitrile), a column temperature of 30°C, an injection volume of 20 μL, a flow rate of 1 mL / min, and a detection wavelength of 245 nm.
[0075] Compare the determination results with the standard and calculate Figure 4 It is the monosaccharide composition of DGSPb-1 of the present invention. Figure 4DGSPb-1 is composed of glucose (Glc), ribose (Rib), rhamnose (Rha), mannose (Man), glucuronic acid (GlcA), galactose (Gal), xylose (Xyl), and arabinose (Ara), with mannose and glucose predominating. The molar ratio of the monosaccharides is Glc:Rib:Rha:Man:GlcA:Gal:Xyl:Ara = 26.97:3.03:0.68:52.44:5.31:3.44:2.68:5.45.
[0076] FT-IR spectrum detection: DGSPb-1 obtained in Example 2 was mixed with potassium bromide in a ratio of 1:100, fully ground and pressed into tablets, and scanned by FT-IR with a scanning wave number range of 4000-400 cm -1 The results are as follows Figure 5 As shown by Figure 5 It can be seen that at 3403.74cm -1 The broad peak at 2925.48 cm represents the stretching vibration of the hydroxyl group OH in DGSPb-1. -1 The peak at 1637.27 cm is the stretching vibration of CH in the -CH2- of the polysaccharide molecule. -1 The absorption peak at 1346.07 cm is the stretching vibration of C=O in -COOR. -1 It is caused by C=H vibration, 1200-1000cm -1 Several absorption peaks at 860 and 920 cm indicate that DGSPb-1 is a pyranose polysaccharide. -1 The absorption peak at indicates that both α- and β-type glycosidic bonds exist in DGSPb-1.
[0077] Nuclear magnetic resonance detection: Weigh 20 mg of DGSPb-1 into a centrifuge tube and add 1 mL of heavy water to dissolve the sample. Take 0.5 mL for 1 H scan analysis, the results are as follows Figure 6 As shown. Generally speaking, δ>5.0ppm ectopic protons are in α configuration, and δ>5.0ppm ectopic protons are in β configuration. Figure 6 The ectopic proton signals of DGSPb-1 are all between 3.3 and 5.3 ppm, indicating that DGSPb-1 contains an α- or β-pyranose ring, which is consistent with the infrared spectroscopy results. The strong absorption peak near 3.5 ppm is the proton peak of the methylene group, and 4.7 ppm is the chemical shift of the solvent D2O.
[0078] Example 4
[0079] This example is the in vitro antioxidant activity determination of the bottom pot water polysaccharide DGSPb-1.
[0080] (1) Determination of DPPH free radical scavenging ability
[0081] The reaction system mixture was reacted at room temperature in the dark for 30 minutes, and the absorbance was measured at 517 nm. The reaction system consisted of 2 mL of DGSPb-1 (2, 4, 6, 8, 10 mg / mL) sample solution and an equal amount of 0.1 mmol / L DPPH ethanol solution. Free radical scavenging rate / % = (1-(A1-A3) / A2) × 100; where A1 is the absorbance of the sample and DPPH solution; A2 is the absorbance of the sample in place of anhydrous ethanol; A3 is the absorbance of the DPPH solution in place of anhydrous ethanol. The measurement results are shown in the figure below. Figure 7 As shown in A, when the concentration of DGSPb-1 is 10 mg / mL, the clearance rate can reach about 80%.
[0082] (2) Determination of total antioxidant capacity
[0083] 0.1 mL DGSPb-1 (2, 4, 6, 8, 10 mg / mL), 1.4 mL freshly prepared FRAP working solution and 2 mL H2O2 were mixed and treated at 37°C for 30 min. The absorbance at A593 nm was measured and the calibration curve was drawn with 0.075-0.00156 μmol / mL FeSO4 as y = 20.248-0.016 (R 2 =0.9997). The results are expressed as FRAP values (Fe 2+ Equivalent) is expressed in (μmol / mL). The results are as follows Figure 7 As shown in B, when the concentration of DGSPb-1 was 10 mg / mL, the FRAP value was 0.0514 μmol / mL.
Claims
1. A combined choline chloride-fructose deep eutectic solvent extraction method for bottom pot water polysaccharides, characterized in that: The following steps are involved: A. After centrifuging the winemaking bottom pot water, the bottom pot water after centrifugation is mixed with a deep eutectic solvent, and liquid-liquid extraction is performed under ultrasonic conditions to obtain an extract; in step A, the deep eutectic solvent is prepared by the following method: after mixing choline chloride and fructose evenly at a molar ratio of 1:1-4, 10-40% of the total mass of choline chloride and fructose is added with water, and heating and stirring are performed until a uniform, stable, clear and transparent liquid is formed, and the extract is obtained; B. Cooling, alcohol precipitation, solid-liquid separation and freeze-drying the extract obtained in step A to obtain crude polysaccharide; C. After the crude polysaccharide of the bottom pot water obtained in step B is prepared into an aqueous solution, Sevage reagent is added to remove the protein, the upper sugar solution is collected, and then soluble small molecules are removed by dialysis. The dialyzed solution is freeze-dried to obtain the bottom pot water polysaccharide DGSP.
2. The combined choline chloride-fructose deep eutectic solvent extraction method of bottom pot water polysaccharide according to claim 1, characterized in that: At least one of the following is met: In step A, the specific operation of standing and filtering the brewing pot water is as follows: after the brewing pot water is stood at 0-4°C for 24-48 hours, it is centrifuged at least once, the first centrifugation conditions are: temperature 0-4°C, speed 8000-10000rpm, time 15-20min; subsequent centrifugation conditions are: temperature 0-4°C, speed 5000-8000rpm, time 10-15min; In step A, the volume ratio of the bottom pot water after centrifugation to the deep eutectic solvent is 1:1-2; In step A, the power of the ultrasound is 200-400W; In step A, the temperature of the liquid-liquid extraction is 40-60° C.; In step A, the liquid-liquid extraction time is 40 to 60 minutes; In step A, when preparing the deep eutectic solvent, the molar ratio of choline chloride to fructose is 1:1-2; In step A, when preparing the deep eutectic solvent, water is added in an amount of 10 to 20% of the total mass of choline chloride and fructose; In step A, when preparing the deep eutectic solvent, the heating and stirring temperature is 80-90° C.; In step A, when preparing the deep eutectic solvent, the heating and stirring time is 5 to 6 hours.
3. The combined choline chloride-fructose deep eutectic solvent extraction method of bottom pot water polysaccharide according to claim 1, characterized in that: At least one of the following is met: In step B, the alcohol precipitation is as follows: the extract and anhydrous ethanol are mixed in a volume ratio of 1:3-5, and allowed to stand at 0-4°C for 24-48 hours; In step B, the solid-liquid separation is carried out by centrifugation; the centrifugation is: centrifuging for 15 to 20 minutes at a temperature of 0 to 4°C and a rotation speed of 8000 to 10000 rpm, and collecting the precipitate; In step B, the freeze-drying conditions are: temperature -80 to -60°C, vacuum degree 1 to 2 Pa, and time 24 to 48 hours.
4. The combined choline chloride-fructose deep eutectic solvent extraction method of bottom pot water polysaccharide according to claim 1, characterized in that: At least one of the following is met: In step C, the crude polysaccharide in the bottom pot water is prepared into an aqueous solution with a concentration of 10 to 20 mg / mL; In step C, the volume ratio of the crude polysaccharide aqueous solution in the bottom pot water to the Sevage reagent is 4 to 5:1; In step C, the upper sugar solution is collected by centrifugation; the centrifugation is: centrifuging at a temperature of 0 to 4°C and a rotation speed of 5000 to 8000 rpm for 10 to 15 minutes to collect the upper sugar solution; In step C, the dialysis is performed by using a dialysis bag at 0 to 4° C. for 36 to 48 hours to retain components with a molecular weight greater than 3500 Da, during which the water is replaced every 6 to 8 hours; In step C, the freeze-drying conditions are: temperature -80 to -60°C, vacuum degree 1 to 2 Pa, and time 24 to 48 hours.
5. The method for extracting bottom pan water polysaccharide by combined choline chloride-fructose deep eutectic solvent according to any one of claims 1 to 4, characterized in that: The following steps are also included: D. After the bottom pot water polysaccharide DGSP obtained in step C is prepared into an aqueous solution, it is first purified by anion exchange column chromatography to obtain bottom pot water polysaccharide DGSPx, and then purified by dextran gel column chromatography to obtain bottom pot water polysaccharide DGSPb-1.
6. The combined choline chloride-fructose deep eutectic solvent extraction method of bottom pot water polysaccharide according to claim 5, characterized in that: At least one of the following is met: In step D, the bottom pot water polysaccharide DGSP obtained in step C is prepared into an aqueous solution with a concentration of 10 to 20 mg / mL; In step D, the anion exchange column chromatography purification operation is: loading the bottom pot water polysaccharide DGSP aqueous solution onto a DEAE52 anion exchange cellulose column, the eluent is sequentially 0-0.4 mol / L NaCl solution, during which the polysaccharide content is determined by the phenol-sulfuric acid method, until no polysaccharide liquid flows out, collecting the effective polysaccharide components at 0 mol / L, 0.1 mol / L and 0.4 mol / L respectively, dialyzing and freeze-drying to obtain the bottom pot water polysaccharide DGSPx, wherein the bottom pot water polysaccharide DGSPx includes DGSPa, DGSPb and / or DGSPc, wherein the effective polysaccharide component at 0 mol / L corresponds to DGSPa, the effective polysaccharide component at 0.1 mol / L corresponds to DGSPb, and the effective polysaccharide component at 0.4 mol / L corresponds to DGSPc; preferably, the eluent is sequentially 0-0.1 mol / L NaCl solution, collecting the effective polysaccharide component at 0.1 mol / L, dialyzing and freeze-drying to obtain DGSPb; In step D, during the anion exchange column chromatography purification, the eluent flow rate is 1-2 mL / min, and every 4-5 mL is collected in one tube.
7. The combined choline chloride-fructose deep eutectic solvent extraction method of bottom pot water polysaccharide according to claim 5, characterized in that: At least one of the following is met: In step D, the DGSPb obtained by purification by anion exchange column chromatography is prepared into an aqueous solution with a concentration of 10 to 20 mg / mL; In step D, the operation of the dextran gel column chromatography purification is as follows: the DGSPb obtained by the anion exchange column chromatography purification is configured into an aqueous solution, loaded onto a Sephadex G-100 dextran gel column chromatography, the eluent is water, during which the polysaccharide content is determined by the phenol-sulfuric acid method, until no polysaccharide liquid flows out, the effective polysaccharide components are collected, dialyzed and freeze-dried to obtain the bottom pot water polysaccharide DGSPb-1; In step D, during the purification by dextran gel column chromatography, the flow rate of the eluent is 1-2 mL / min, and every 4-5 mL is collected in one tube.
8. The method for extracting bottom pan water polysaccharide by combined choline chloride-fructose deep eutectic solvent according to any one of claims 5 to 7, characterized in that: In step D, the molecular weight of the bottom pot water polysaccharide DGSPb-1 is 11.8832 kDa, and the molar ratio of its monosaccharide composition is Glc:Rib:Rha:Man:GlcA:Gal:Xyl:Ara=26.97:3.03:0.68:52.44:5.31:3.44:2.68:5.
45.
9. The bottom pot water polysaccharide DGSP extracted by the method described in any one of claims 1 to 4, the bottom pot water polysaccharide DGSPx extracted by the method described in any one of claims 5 to 6, and / or the bottom pot water polysaccharide DGSPb-1 extracted by the method described in any one of claims 5 to 8.
10. Use of the bottom pot water polysaccharide DGSP extracted by the method described in any one of claims 1 to 4, the bottom pot water polysaccharide DGSPx extracted by the method described in any one of claims 5 to 6, and / or the bottom pot water polysaccharide DGSPb-1 extracted by the method described in any one of claims 5 to 8 in the preparation of free radical scavengers; preferably, the free radicals are DPPH free radicals and / or OH free radicals.