Pot-bottom water polysaccharide as well as deep eutectic solvent extraction method and application of pot-bottom water polysaccharide

Abstract

The invention discloses a pot-bottom water polysaccharide as well as a deep eutectic solvent extraction method and application thereof, and belongs to the technical field of active substance extraction. In order to realize efficient and green extraction of the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the polysaccharose from the deep eutectic solvent is prepared from choline chloride, oxalic acid, fructose and water. The ternary deep eutectic solvent system is researched, the extraction rate of the pot-bottom water polysaccharide is increased to 45% or above, the free radical scavenging capacity of the pot-bottom water polysaccharide is remarkably improved, and the extraction rate and the antioxidant activity of the pot-bottom water polysaccharide are remarkably superior to those of a traditional hot water extraction process and other deep eutectic solvent systems.

Description

Technical Field

[0001] The invention belongs to the technical field of active substance extraction, and particularly relates to a bottom pot water polysaccharide and a deep eutectic solvent extraction method and application thereof. Background Art

[0002] In recent years, my country's liquor economy has developed rapidly, and wastewater is also produced in large quantities during the liquor brewing process, which is about 12 times the alcohol output. The bottom pot water of liquor brewing wastewater is a mixed water slurry with mash, tail, residual dregs, yellow water, etc. left at the bottom of the pot after distillation. It has the characteristics of low pH value, high chroma, and high organic pollutants. In addition, the bottom pot water has a complex composition, 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 also lead to the organic components not being fully utilized, resulting in resource waste. Therefore, the resource utilization of the bottom pot water is of great significance.

[0003] With the rise of the concept of healthy diet, the extraction of natural active ingredients for functional foods, medicines, etc. has received widespread attention. Polysaccharides are a type 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 blood lipid reduction, and are widely used in clinical treatment and prevention of diseases. At present, the resource utilization of bottom pot water in liquor brewing mainly focuses on the production of bacteria, the preparation of fragrance liquid and the preparation of organic fertilizer, such as CN116769840A discloses the production of Chlorella growth factor using bottom pot water. CN115160030A uses bottom pot water to produce organic fertilizer containing γ-polyglutamic acid; CN101897271A discloses a method for producing bald puffball mycelium using bottom pot water; CN113755537A uses bottom pot water to prepare butyric acid. In terms of the preparation of natural polysaccharides, only CN117699876A discloses the extraction of crude polysaccharides from bottom pot water.

[0004] At present, the extraction of natural polysaccharides mostly adopts assisted ultrasound, microwave and enzyme methods, and the extraction solvents are mostly water and organic solvents. The organic solvent extraction solvent residue is not green and economical, and the subsequent separation is difficult. Water as a solvent has a low extraction rate and high energy consumption in the high-temperature extraction process. Deep eutectic solvent (DES) is a new type of green extractant formed by mixing and heating hydrogen acceptors and hydrogen donors in a suitable proportion. It has the advantages of simple operation, non-toxicity, green environmental protection, low cost, recyclability, and high extraction efficiency. In recent years, the application of DES in the extraction of polysaccharide components has gradually gained favor. For example, Jing et al. (Effect of deep eutectic solvent extraction on Auricularia auricula polysaccharide solubilization and antioxidant potential [J]. Sustainable Chemistry and Pharmacy, 2023, 34: 101166) used DES solvent (ethanol-choline chloride) to extract Auricularia auricula polysaccharide. Compared with the hot water extraction method, the yield of Auricularia auricula polysaccharide increased by 18.68%; He Xianyu et al. (Study on the extraction process of Ganoderma lucidum polysaccharide with choline chloride / lactic acid / guaiacol ternary eutectic solvent and its antioxidant activity [J]. Food Industry Science and Technology, 2024, 45 (14): 34-41) found that the yield of Ganoderma lucidum polysaccharide extracted by DES with a water content of 50% (9.20% ± 0.27%) was 4.3 times that of the hot water method (2.14% ± 0.08%). Ye Zhiqiang et al. (Ye Zhiqiang. Separation, purification, structural identification and activity evaluation of shiitake mushroom polysaccharides extracted by deep eutectic solvent-enhanced subcritical water [D]. Yangzhou University, 2023.) used deep eutectic solvent-enhanced subcritical water to extract polysaccharides from shiitake mushrooms. Under optimal conditions, the extraction rate of polysaccharides was increased by 19.24% and 17.01% compared with subcritical water extraction and hot water extraction, respectively.

[0005] However, deep eutectic solvent extraction is affected by many factors, such as temperature, time, water content and combination system, among which the deep eutectic solvent combination system has the greatest impact. For different raw materials, different deep eutectic solvent combination systems have significant differences in extraction effects. In addition, the antioxidant activity of polysaccharides prepared by different extraction methods and deep eutectic solvent combination systems also varies greatly. Therefore, it is necessary to screen the most suitable deep eutectic solvent combination system for different extraction objects.

[0006] Therefore, how to improve the extraction effect of bottom pot water polysaccharides, obtain an efficient extraction method for bottom pot water polysaccharides, and ensure that the bottom pot water polysaccharides have excellent antioxidant activity are technical problems that need to be solved in this field. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for extracting polysaccharides from water chestnuts with a deep eutectic solvent with a high yield. The yield of polysaccharides extracted from water chestnuts by this method reaches more than 45%, which solves the gap in the existing high-efficiency and green extraction technology of polysaccharides from water chestnuts. At the same time, polysaccharides from water chestnuts have excellent antioxidant activity.

[0008] To achieve the above object, the present invention first provides a deep eutectic solvent extraction method for bottom pot water polysaccharide, which comprises the following steps:

[0009] A. After centrifuging the brewing pot water, the centrifuged pot water is mixed with a deep eutectic solvent, and liquid-liquid extraction is performed under ultrasonic conditions to obtain an extract;

[0010] B. Cooling the extract obtained in step A, precipitating with alcohol, collecting the precipitate and freeze-drying it to obtain crude polysaccharide from bottom pot water;

[0011] 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 bottom pot water polysaccharide solution is collected, and freeze-dried to obtain the bottom pot water polysaccharide;

[0012] In step A, the deep eutectic solvent is prepared by the following method: choline chloride, oxalic acid and fructose are taken in a molar ratio of 1 to 2: 1 to 2: 1 to 4, mixed evenly, and then 10 to 40% of the total mass of choline chloride, oxalic acid and fructose is added with water, and heated and stirred (to promote the formation of hydrogen bonds between the components) until a uniform, stable, clear and transparent liquid is formed.

[0013] Preferably, in the above-mentioned deep eutectic solvent extraction method of bottom pot water polysaccharide, in step A, the volume ratio of the bottom pot water after centrifugation to the deep eutectic solvent is 1:0.5-1.5.

[0014] More preferably, in the above-mentioned method for extracting polysaccharides from bottom pot water with a deep eutectic solvent, in step A, the volume ratio of the bottom pot water after centrifugation to the deep eutectic solvent is 1:0.5-1.

[0015] Among them, in the deep eutectic solvent extraction method of the above-mentioned bottom pot water polysaccharide, in step A, the specific operation of standing and centrifuging 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.

[0016] Wherein, in the above-mentioned deep eutectic solvent extraction method of bottom pot water polysaccharide, in step A, the power of the ultrasound is 200-400W.

[0017] Among them, in the deep eutectic solvent extraction method of the bottom pot water polysaccharide, in step A, the temperature of the liquid-liquid extraction is 40-60°C.

[0018] Among them, in the deep eutectic solvent extraction method of the bottom pot water polysaccharide, in step A, the time of the liquid-liquid extraction is 40 to 60 minutes.

[0019] Preferably, in the above-mentioned deep eutectic solvent extraction method for polysaccharides from bottom pot water, in step A, when preparing the deep eutectic solvent, the molar ratio of choline chloride, oxalic acid and fructose is 1-2:1-2:1-4.

[0020] More preferably, in the above-mentioned deep eutectic solvent extraction method for polysaccharides from bottom pot water, in step A, when preparing the deep eutectic solvent, the molar ratio of choline chloride, oxalic acid and fructose is 1:1:1-2.

[0021] Preferably, in the above-mentioned deep eutectic solvent extraction method for bottom pot water polysaccharide, in step A, when preparing the deep eutectic solvent, water is added in an amount of 20 to 40% of the total mass of choline chloride, oxalic acid and fructose.

[0022] Wherein, in the above-mentioned deep eutectic solvent extraction method of bottom pot water polysaccharide, in step A, when preparing the deep eutectic solvent, the heating and stirring temperature is 80-90°C.

[0023] Wherein, in the above-mentioned deep eutectic solvent extraction method of bottom pot water polysaccharide, in step A, when preparing the deep eutectic solvent, the heating and stirring time is 5 to 6 hours.

[0024] Among them, in the deep eutectic solvent extraction method of the above-mentioned bottom pot water polysaccharide, 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 and precipitate at 0-4°C for 24-48 hours.

[0025] Wherein, in the above-mentioned deep eutectic solvent extraction method of bottom pot water polysaccharide, in step B, the precipitate is collected by centrifugation.

[0026] Preferably, in the deep eutectic solvent extraction method of the above-mentioned bottom pot water polysaccharide, in step B, when collecting the precipitate, 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 to collect the precipitate.

[0027] Among them, in the deep eutectic solvent extraction method of the bottom pot water polysaccharide, in step B, the freeze-drying conditions are: temperature -80 to -60°C, vacuum degree 1 to 2Pa, and time 24 to 48h.

[0028] Among them, in the deep eutectic solvent extraction method of the bottom pot water polysaccharide, in step C, the crude bottom pot water polysaccharide is prepared into an aqueous solution with a concentration of 10 to 25 mg / mL.

[0029] Among them, in the above-mentioned deep eutectic solvent extraction method of bottom pot water polysaccharide, in step C, the volume ratio of the bottom pot water crude polysaccharide aqueous solution and the Sevage reagent is 4 to 5:1.

[0030] Wherein, in the above-mentioned deep eutectic solvent extraction method of bottom pot water polysaccharide, in step C, the bottom pot water polysaccharide solution is collected by centrifugation.

[0031] Preferably, in the above-mentioned deep eutectic solvent extraction method of bottom pot water polysaccharide, in step C, when collecting the bottom pot water polysaccharide solution, the centrifugation is: centrifuging for 10 to 15 minutes at a temperature of 0 to 4°C and a rotation speed of 5000 to 8000 rpm, and collecting the supernatant.

[0032] Among them, in the deep eutectic solvent extraction method of the bottom pot water polysaccharide, in step C, the freeze-drying conditions are: temperature -80 to -60°C, vacuum degree 1 to 2Pa, and time 24 to 48h.

[0033] The present invention also provides a bottom pot water polysaccharide, which is extracted by using the deep eutectic solvent extraction method of the bottom pot water polysaccharide.

[0034] The present invention also provides the use of the bottom pot water polysaccharide in preparing a free radical scavenger.

[0035] The bottom pot water polysaccharide obtained by the present invention has excellent performance in scavenging DPPH free radicals and ABTS free radicals. 50 The values ​​are about 0.13 and 0.35 mg / mL respectively. Therefore, in the above-mentioned use, the free radical is preferably a DPPH free radical or an ABTS free radical.

[0036] In the present invention, the bottom boiler water of liquor brewing wastewater is a well-known substance in the art, which can refer to literature 1 (Luo Yuting, Zhang Yuliang, Shui Liangyang, etc. Research on the treatment of bottom boiler water of liquor brewing wastewater with quadriceps sp. [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.

[0037] Beneficial effects of the present invention:

[0038] In view of the complex and diverse characteristics of bottom pot water components, the present invention has conducted a lot of exploration on the ternary deep eutectic solvent combination system, and the extraction rate of bottom pot water polysaccharides has been increased to more than 45%, which is more than 3.37 times that of the traditional hot water extraction process, and is also significantly better than other deep eutectic solvent combination systems. The bottom pot water polysaccharide substances can be more fully extracted. In addition, the method of the present invention has mild extraction conditions, a simple extraction process, easy operation, no reliance on high-end precision instruments, and low cost.

[0039] The bottom pot water polysaccharide obtained by the present invention exhibits beneficial antioxidant activity, and the scavenging capacity of DPPH free radical and ABTS free radical is IC 50 The values ​​reached about 0.13 and 0.35 mg / mL respectively, which are significantly better than the traditional hot water extraction process and other deep eutectic solvent combination systems. They can be used to prepare free radical scavengers and provide a theoretical basis for the in-depth development of bottom pot water. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The present invention is a flow chart of the method for separating bottom pan water polysaccharides.

[0041] Figure 2 This is a scanning electron microscope image of the bottom pot water polysaccharide obtained in Example 1.

[0042] Figure 3 This is a scanning electron micrograph of the bottom pot water polysaccharide obtained in Comparative Example 11. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below by way of examples, but the protection scope of the present invention is not limited to the scope of the examples.

[0044] Example 1

[0045] (1) After the brewing pot water is left to stand at 4°C for 24 hours, it is centrifuged at 4°C and 10,000 rpm for 15 minutes. This process is repeated twice with the same centrifugation parameters. The pot water after centrifugation can be stored at 4°C for a long time.

[0046] (2) Take choline chloride, oxalic acid and fructose in a molar ratio of 1:1:1, then add 20% of the total mass of water to the mixture, heat and stir at 80°C until a uniform, stable, clear and transparent liquid is formed, thereby obtaining a deep eutectic solvent; take 50 mL of the bottom pot water after centrifugation and 50 mL of the deep eutectic solvent and mix them evenly, and perform liquid-liquid extraction at 50°C at a power of 200 W in an ultrasonic extractor for 40 min.

[0047] (3) After the extraction is completed, the mixed liquid of the bottom pot water and the deep eutectic solvent is taken out and cooled to room temperature, and then 5 times the volume of anhydrous ethanol is added and allowed to stand for precipitation at 4°C for 36 hours. After the alcohol precipitation is completed, the precipitate is collected by centrifugation and freeze-dried at -80°C and a vacuum degree of about 1Pa for 48 hours to obtain the crude bottom pot water polysaccharide.

[0048] (4) Take 1 g of the crude polysaccharide sample of the bottom pot water, add 100 mL of deionized water to prepare a 10 mg / mL solution, and then add 20 mL of Sevage reagent (Sevage reagent is a mixture of dichloromethane and n-butanol in a volume ratio of 4:1). After mixing evenly, centrifuge at 4°C and 10,000 rpm for 15 min to remove the Sevage reagent, collect the bottom pot water polysaccharide solution, and then freeze-dry to obtain the bottom pot water polysaccharide sample, and determine the polysaccharide content by the phenol-sulfuric acid method.

[0049] Example 2

[0050] (1) After the brewing pot water is left to stand at 4°C for 24 hours, it is centrifuged at 4°C and 10,000 rpm for 15 minutes. This process is repeated twice with the same centrifugation parameters. The pot water after centrifugation can be stored at 4°C for a long time.

[0051] (2) Take choline chloride, oxalic acid and fructose in a molar ratio of 1:1:2, then add 20% of the total mass of water, heat and stir at 90°C until a uniform, stable, clear and transparent liquid is formed, thereby obtaining a deep eutectic solvent; take 60 mL of the bottom pot water after centrifugation and 40 mL of the deep eutectic solvent and mix them evenly, and perform liquid-liquid extraction in an ultrasonic extractor (200 W) at 50°C for 40 min.

[0052] (3) After the extraction is completed, the mixed liquid of the bottom pot water and the deep eutectic solvent is taken out and cooled to room temperature, and then 5 times the volume of anhydrous ethanol is added and allowed to stand for precipitation at 4°C for 36 hours. After the alcohol precipitation is completed, the precipitate is collected by centrifugation and freeze-dried for 48 hours at -80°C and a vacuum degree of about 1Pa to obtain the crude bottom pot water polysaccharide.

[0053] (4) Take 1 g of the crude polysaccharide sample of the bottom pot water, add 100 mL of deionized water to prepare a 10 mg / mL solution, and then add 20 mL of Sevage reagent (Sevage reagent is a mixture of dichloromethane and n-butanol in a volume ratio of 4:1). After mixing evenly, centrifuge at 4°C and 10,000 rpm for 15 min to remove the Sevage reagent, collect the bottom pot water polysaccharide solution, and then freeze-dry to obtain the bottom pot water polysaccharide sample, and calculate its yield and polysaccharide content.

[0054] Example 3

[0055] (1) After the brewing pot water is left to stand at 4°C for 24 hours, it is centrifuged at 4°C and 10,000 rpm for 15 minutes. This process is repeated twice with the same centrifugation parameters. The pot water after centrifugation can be stored at 4°C for a long time.

[0056] (2) Take choline chloride, oxalic acid and fructose in a molar ratio of 1:1:1, then add 20% of the total mass of water to the mixture, heat and stir at 90°C until a uniform, stable, clear and transparent liquid is formed to obtain a deep eutectic solvent; take 60 mL of the bottom pot water after centrifugation and 30 mL of the deep eutectic solvent and mix them evenly, and perform liquid-liquid extraction at 50°C at a power of 200 W for 40 min.

[0057] (3) After the extraction is completed, the mixed liquid of the bottom pot water and the deep eutectic solvent is taken out and cooled to room temperature, and then 5 times the volume of anhydrous ethanol is added and allowed to stand for precipitation at 4°C for 36 hours. After the alcohol precipitation is completed, the precipitate is collected by centrifugation and freeze-dried for 48 hours at -80°C and a vacuum degree of about 1Pa to obtain the crude bottom pot water polysaccharide.

[0058] (4) Take 1 g of the crude polysaccharide sample of the bottom pot water, add 100 mL of deionized water to prepare a 10 mg / mL solution, and then add 20 mL of Sevage reagent (Sevage reagent is a mixture of dichloromethane and n-butanol in a volume ratio of 4:1). After mixing evenly, centrifuge at 4°C and 10,000 rpm for 15 min to remove the Sevage reagent, collect the bottom pot water polysaccharide solution, and then freeze-dry to obtain the bottom pot water polysaccharide sample, and calculate its yield and polysaccharide content.

[0059] Comparative Example 1

[0060] The process is the same as Example 3 except that the deep eutectic solvent is prepared by combining choline chloride and fructose in a molar ratio of 1:2 (water 20%).

[0061] Comparative Example 2

[0062] The process is the same as that in Example 3 except that the deep eutectic solvent is prepared by combining choline chloride and fructose in a molar ratio of 1:1 (water 20%).

[0063] Comparative Example 3

[0064] The process is the same as that of Example 3 except that the deep eutectic solvent is prepared by combining choline chloride and oxalic acid in a molar ratio of 1:1 (water 20%).

[0065] Comparative Example 4

[0066] The process is the same as Example 3 except that the deep eutectic solvent is prepared by combining choline chloride and malic acid in a molar ratio of 1:1 (water 20%).

[0067] Comparative Example 5

[0068] The process is the same as that of Example 3 except that the deep eutectic solvent is prepared by combining choline chloride and urea in a molar ratio of 1:1 (water 20%).

[0069] Comparative Example 6

[0070] The process is the same as that of Example 3 except that the deep eutectic solvent is prepared by combining choline chloride and glycerol in a molar ratio of 1:1 (water 20%).

[0071] Comparative Example 7

[0072] The process is the same as that of Example 3 except that the deep eutectic solvent is prepared by combining choline chloride and sorbitol in a molar ratio of 1:1 (water 20%).

[0073] Comparative Example 8

[0074] The process is the same as that of Example 3 except that the deep eutectic solvent is prepared by combining choline chloride and malonic acid in a molar ratio of 1:1 (water 20%).

[0075] Comparative Example 9

[0076] Except that the volume ratio of the bottom pot water after centrifugation to the deep eutectic solvent is 1:2 (30 mL of the bottom pot water after centrifugation and 60 mL of the deep eutectic solvent), the rest is the same as Example 3.

[0077] Comparative Example 10

[0078] Except that the volume ratio of the bottom pot water after centrifugation to the deep eutectic solvent is 1:0.2 (50 mL of the bottom pot water after centrifugation and 10 mL of the deep eutectic solvent), the rest is the same as Example 3.

[0079] Comparative Example 11

[0080] The crude polysaccharide sample of the bottom pot water was prepared by the traditional hot water extraction method. 60 mL of the pretreated bottom pot water was directly taken, and 30 mL of 40° C. deionized water was added for extraction. Other conditions were the same as those in Example 3.

[0081] Experimental Example 1

[0082] The yield of polysaccharide in bottom water was measured for Examples 1 to 3 and Comparative Examples 1 to 11. The results are shown in Table 1.

[0083] Table 1 Yield of polysaccharides from different treatments of bottom pot water

[0084] Group Extraction yield (mg / mL) purity(%) Group Extraction yield (mg / mL) purity(%) Example 1 46.34 57.00 Comparative Example 5 24.02 29.73 Example 2 45.83 56.90 Comparative Example 6 24.00 35.86 Example 3 49.00 56.08 Comparative Example 7 26.34 38.276 Comparative Example 1 25.63 37.00 Comparative Example 8 23.19 35.696 Comparative Example 2 24.32 35.54 Comparative Example 9 31.26 34.26 Comparative Example 3 23.25 33.54 Comparative Example 10 29.76 31.11 Comparative Example 4 22.48 34.04 Comparative Example 11 14.55 20.14

[0085] Note: The extraction yield is expressed as mg of polysaccharide extracted from 1 mL of centrifuged bottom pot water; the purity is expressed as the percentage of polysaccharide in the bottom pot water polysaccharide.

[0086] After the exploration of the present invention, the yield of bottom pot water polysaccharides extracted by the binary deep eutectic solvent combination (Comparative Examples 1 to 10) is between 22% and 31%, which is relatively improved compared to hot water extraction (14.55%), but the extraction efficiency needs to be further improved. Due to the complex and diverse components of bottom pot water, the polysaccharide components therein are not fully extracted, which obviously causes a waste of resources. Therefore, the present invention continues to conduct a lot of exploration on the ternary deep eutectic solvent combination system. After adopting the extraction scheme of the present invention, the extraction rate can be increased to about 45%, which is 3.37 times that of hot water extraction, and is also significantly better than other deep eutectic solvent combination systems, and can fully extract bottom pot water polysaccharides.

[0087] Experimental Example 2

[0088] The antioxidant activity (ability to scavenge DPPH free radicals and ABTS free radicals) of the bottom pot water polysaccharides obtained in Examples 1 to 3 and Comparative Examples 1 to 11 was measured.

[0089] Determination of the ability to scavenge DPPH free radicals: Take the bottom pot water polysaccharides obtained in Examples 1 to 3 and Comparative Examples 1 to 11 and prepare them into 0.02-2 mg / mL solutions as test samples. Take 2 mL of the test sample and an equal amount of 0.1 mmol / L DPPH ethanol solution and place them at room temperature in the dark for 30 minutes, then measure the absorbance at 517 nm. Free radical scavenging rate / % = (1-(A1-A3) / A2) × 100; wherein A1 is the absorbance of the sample and the DPPH solution; A2 is the absorbance of the sample replaced by anhydrous ethanol; A3 is the absorbance of the DPPH solution replaced by anhydrous ethanol. The test results are shown in Table 2.

[0090] Determination of the ability to scavenge ABTS free radicals: Take the bottom pot water polysaccharides obtained in Examples 1 to 3 and Comparative Examples 1 to 11 and prepare them into a solution of 0.02 to 2 mg / mL as the sample to be tested. Take 1 mL of the sample to be tested and 3 mL of ABTS working solution and shake them evenly. After reacting for 30 minutes at room temperature and away from light, measure the absorbance value (A1) at 734 nm. Anhydrous ethanol is used instead of the sample to measure the absorbance value A2; anhydrous ethanol is used instead of ABTS solution to measure the absorbance value A3. Free radical scavenging rate / % = (1-(A1-A3) / A2)×100. The measurement results are shown in Table 2.

[0091] Table 2 Antioxidant capacity of polysaccharides in different treatments of bottom pot water

[0092]

[0093] As shown in Table 2, the polysaccharide obtained from the present invention exhibits beneficial antioxidant activity, and its scavenging capacity for DPPH free radicals and ABTS free radicals is IC 50The values ​​reached about 0.13 and 0.35 mg / mL respectively, which are significantly better than the traditional hot water extraction process and other deep eutectic solvent combination systems.

[0094] Experimental Example 3

[0095] The embodiment 1 and the comparative example 11 were subjected to electron microscope scanning to analyze their microstructures. Figure 2 It shows that the bottom pot water polysaccharide obtained in Example 1 has a smooth surface and a flaky structure. Figure 3 It is shown that the surface of the bottom pot water polysaccharide obtained in Comparative Example 11 is dense and granular.

Claims

1. A deep eutectic solvent extraction method for bottom pot water polysaccharides, characterized in that: The following steps are involved: A. After centrifuging the brewing pot water, the centrifuged pot water is mixed with a deep eutectic solvent, and liquid-liquid extraction is performed under ultrasonic conditions to obtain an extract; B. Cooling the extract obtained in step A, precipitating with alcohol, collecting the precipitate and freeze-drying it to obtain crude polysaccharide from bottom pot water; 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 bottom pot water polysaccharide solution is collected, and freeze-dried to obtain the bottom pot water polysaccharide; In step A, the deep eutectic solvent is prepared by the following method: choline chloride, oxalic acid and fructose are taken in a molar ratio of 1-2:1-2:1-4, mixed evenly, and then 10-40% of the total mass of water of choline chloride, oxalic acid and fructose is added, and heated and stirred until a uniform, stable, clear and transparent liquid is formed.

2. The deep eutectic solvent extraction method of bottom pan water polysaccharide according to claim 1, characterized in that: In step A, the volume ratio of the bottom pot water after centrifugation to the deep eutectic solvent is 1:0.5-1.5; preferably 1:0.5-1.

3. The deep eutectic solvent extraction method of bottom pan water polysaccharide according to claim 1, characterized in that: At least one of the following is met: In step A, the specific operation of centrifuging the brewing bottom pot water is as follows: after the brewing bottom pot water is allowed to stand 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 are: temperature 0-4°C, speed 5000-8000rpm, time 10-15min; 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, oxalic acid and fructose is 1-2:1-2:1-4; preferably 1:1:1-2; In step A, when preparing the deep eutectic solvent, water is added in an amount of 20 to 40% of the total mass of choline chloride, oxalic acid 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.

4. The deep eutectic solvent extraction method of bottom pan 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 precipitate is collected by centrifugation; the centrifugation is: centrifugation for 15 to 20 minutes at a temperature of 0 to 4°C and a rotation speed of 8000 to 10000 rpm to collect 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.

5. The deep eutectic solvent extraction method of bottom pan water polysaccharide according to claim 1, characterized in that: In step C, the crude polysaccharide from the bottom pot water is prepared into an aqueous solution with a concentration of 10 to 25 mg / mL.

6. The deep eutectic solvent extraction method of bottom pan water polysaccharide according to claim 1, characterized in that: In step C, the volume ratio of the crude polysaccharide aqueous solution in the bottom pot water to the Sevage reagent is 4-5:

1.

7. The deep eutectic solvent extraction method of bottom pan water polysaccharide according to claim 1, characterized in that: In step C, the polysaccharide solution in the bottom pot water is collected by centrifugation; the centrifugation is: centrifuging for 10 to 15 minutes at a temperature of 0 to 4°C and a rotation speed of 5000 to 8000 rpm, and collecting the supernatant.

8. The deep eutectic solvent extraction method of bottom pan water polysaccharide according to any one of claims 1 to 7, characterized in that: 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.

9. The bottom pot water polysaccharide obtained by extraction using the method described in any one of claims 1 to 8.

10. Use of the bottom pot water polysaccharide extracted by the method according to any one of claims 1 to 8 in the preparation of a free radical scavenger; preferably, the free radical is a DPPH free radical or an ABTS free radical.

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