WO2024104083A1 - Extraction method for small-molecule corn bran polysaccharide - Google Patents
Extraction method for small-molecule corn bran polysaccharide Download PDFInfo
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 154
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 154
- 150000004676 glycans Chemical class 0.000 title claims abstract description 149
- 240000008042 Zea mays Species 0.000 title claims abstract description 90
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 131
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- 238000000034 method Methods 0.000 claims abstract description 33
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- 238000001556 precipitation Methods 0.000 claims abstract description 24
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- 239000004310 lactic acid Substances 0.000 claims abstract description 11
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
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- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Substances ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 claims description 3
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
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Abstract
Disclosed in the present invention is an extraction method for a small-molecule corn bran polysaccharide. The method comprises: crushing and sieving corn bran; subjecting the sieved corn bran to heating extraction with an acidic eutectic solvent, adding water thereto, stirring the resulting mixture until same is uniformly dispersed, and then filtering same for separation; subjecting the resulting filtrate to rotary evaporation to remove water molecules, so as to obtain a concentrated solution of a polysaccharide in the eutectic solvent, adding absolute ethyl alcohol thereto, leaving the resulting mixture to stand, and subjecting same to centrifugal separation, washing same with absolute ethyl alcohol and freeze-drying, so as to obtain a crude corn bran polysaccharide; and purifying the crude corn bran polysaccharide by means of gradient alcohol precipitation with ethanol, so as to obtain a small-molecule corn bran polysaccharide. In the technical solution of the present invention, the corn bran polysaccharide extracted by using an acidic eutectic solvent has a low molecular weight, and the process is simple. In the invention, the crude corn bran polysaccharide extracted by preferably using a lactic acid/choline chloride eutectic solvent has a high extraction rate, and the molar ratio of lactic acid to choline chloride is 10 : 1, which reduces the process cost.
Description
本发明涉及多糖提取技术领域，具体的说是涉及一种玉米皮小分子多糖的提取方法。The invention relates to the technical field of polysaccharide extraction, in particular to a method for extracting corn husk small-molecule polysaccharides.
玉米是目前世界种植面积最广的农作物品种，全球年产量约为10.9亿吨，中国的玉米生产量位居世界第二，国家***在2020年统计，全国玉米的播种面积为4126.4万公顷，单位面积产量6317公斤/公顷，总产量为2.606亿吨，约占全球玉米产量的20％以上。玉米皮主要由玉米籽粒最外层的果皮组织组成，在我国，玉米皮是玉米淀粉工业湿法碾磨过程中的主要副产品，约占玉米干质量的10％-14％，产量十分巨大。经国家权威机构药理、药效研究证明：活性多糖具有快速有效地激活、增强和调节人体免疫能力，抵制肿瘤、抗炎、抗病毒、调整血糖血脂、修复自身免疫性疾病等神奇功效。玉米皮活性多糖可以降低血清胆固醇、预防高血脂和肥胖症、促进中毒性物质的排除从而减少直肠癌的发生，被称为是“第七营养素”。倘若能进一步利用玉米深加工副产物玉米皮开发玉米皮多糖，既可大大提高玉米加工企业的经济效益，同时也是获得玉米皮活性多糖产品开发提供一条有效途径。Corn is currently the most widely planted crop variety in the world, with an annual global output of about 1.09 billion tons. China's corn production ranks second in the world. According to statistics from the National Bureau of Statistics in 2020, the national corn planting area is 41.264 million hectares, the unit area yield is 6,317 kg/hectare, and the total output is 260.6 million tons, accounting for more than 20% of the global corn production. Corn husk is mainly composed of the outermost pericarp tissue of corn kernels. In my country, corn husk is the main by-product of the industrial wet milling process of corn starch, accounting for about 10%-14% of the dry mass of corn, and the output is very huge. Pharmacological and efficacy studies by national authoritative institutions have proved that active polysaccharides have the magical effects of quickly and effectively activating, enhancing and regulating human immunity, resisting tumors, anti-inflammatory, anti-viral, adjusting blood sugar and blood lipids, and repairing autoimmune diseases. Corn husk active polysaccharides can lower serum cholesterol, prevent hyperlipidemia and obesity, promote the elimination of toxic substances, and thus reduce the occurrence of rectal cancer. It is called the "seventh nutrient." If we can further utilize corn husk, a by-product of corn deep processing, to develop corn husk polysaccharides, it will not only greatly improve the economic benefits of corn processing companies, but also provide an effective way to develop active corn husk polysaccharide products.
目前对多糖的提取方法主要是主要有水提醇沉提取，超声波提取、微波提取、酶提取等，现有提取方法中的提取率较低，且酶提取方法成本高、工艺条件要求严格。At present, the main extraction methods for polysaccharides include water extraction, alcohol precipitation extraction, ultrasonic extraction, microwave extraction, enzyme extraction, etc. The extraction rate of the existing extraction methods is low, and the enzyme extraction method is costly and requires strict process conditions.
因此，提供一种提取率高、成本低、适合工业化生产的玉米皮小分子多糖的提取方法是本领域技术人员亟需解决的技术问题Therefore, providing a method for extracting corn bran small molecule polysaccharides with high extraction rate, low cost and suitable for industrial production is a technical problem that technicians in this field need to solve urgently.
发明内容
Summary of the invention
为解决现有技术中玉米皮多糖提取率较低、提取的多糖分子量大、提取方法成本高、工艺条件要求严格的缺陷，本发明提供一种提取成本低、工艺简单、提取率高的玉米皮小分子多糖的提取方法。In order to solve the defects in the prior art of low corn bran polysaccharide extraction rate, large molecular weight of extracted polysaccharides, high extraction method cost and strict process conditions, the present invention provides a method for extracting corn bran small molecule polysaccharides with low extraction cost, simple process and high extraction rate.
为了实现上述目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solution:
一种小分子玉米皮多糖的提取方法，包括以下步骤：A method for extracting small molecule corn bran polysaccharides comprises the following steps:
(1)取玉米皮进行粉碎，然后过筛；(1) taking corn husks, crushing them, and then sieving them;
(2)使用酸性低共熔溶剂对过筛的玉米皮进行加热浸提，结束后加入低共熔溶剂3-8倍体积的水搅拌分散均匀之后，过滤分离；(2) using an acidic deep eutectic solvent to heat and extract the sieved corn husk, adding 3-8 times the volume of the deep eutectic solvent of water after stirring and dispersing evenly, and filtering and separating;
(3)将过滤液旋蒸去除水分子得到多糖低共熔溶剂浓缩液，向浓缩液中加入浓缩液4-5倍体积的无水乙醇，1-4℃下存放静置，离心分离，经无水乙醇洗涤，冷冻干燥得到玉米皮粗多糖；(3) removing water molecules from the filtrate by rotary evaporation to obtain a polysaccharide deep eutectic solvent concentrate, adding 4-5 times the volume of anhydrous ethanol to the concentrate, storing at 1-4° C., centrifuging, washing with anhydrous ethanol, and freeze-drying to obtain corn husk crude polysaccharide;
(4)将玉米皮粗多糖经过乙醇分级醇沉纯化得到小分子玉米皮多糖。(4) The crude corn husk polysaccharide is purified by ethanol graded precipitation to obtain small molecular weight corn husk polysaccharide.
进一步，步骤(1)中所述过筛粒度为40-60目。Furthermore, the sieved particle size in step (1) is 40-60 mesh.
进一步，步骤(2)中所述酸性低共熔溶剂为甲酸/氯化胆碱、草酸/氯化胆碱、甲酸/乙酸/氯化胆碱、乳酸/氯化胆碱中的任一种。Furthermore, the acidic low eutectic solvent in step (2) is any one of formic acid/choline chloride, oxalic acid/choline chloride, formic acid/acetic acid/choline chloride, and lactic acid/choline chloride.
优选的，所述酸性低共熔溶剂为乳酸/氯化胆碱；Preferably, the acidic deep eutectic solvent is lactic acid/choline chloride;
更进一步，所述酸性低共熔溶剂中酸与氯化胆碱的摩尔比为(2-10)：1。Furthermore, the molar ratio of acid to choline chloride in the acidic deep eutectic solvent is (2-10):1.
采用上述进一步方案的有益效果在于：本发明采用的氯化胆碱/乳酸DESs对木质素具有高溶解度，而对纤维素基本不溶。当乳酸的摩尔比增大时，木质素的溶解度也随之提高，同时对玉米皮的致密结构破坏严重，更有利于玉米皮多糖的提取以及小分子结构的形成。The beneficial effect of adopting the above further scheme is that the choline chloride/lactic acid DESs used in the present invention has high solubility for lignin, but is basically insoluble in cellulose. When the molar ratio of lactic acid increases, the solubility of lignin also increases, and at the same time, the dense structure of corn husk is severely damaged, which is more conducive to the extraction of corn husk polysaccharides and the formation of small molecular structures.
进一步，步骤(2)中所述加热浸提的温度为100-140℃，加热时间为0.5-3h；Furthermore, the temperature of the heating and leaching in step (2) is 100-140° C., and the heating time is 0.5-3 h;
所述加入水的温度为80-100℃，搅拌速率为60-300rmp，搅拌时间为10-20min。
The temperature of adding water is 80-100° C., the stirring rate is 60-300 rpm, and the stirring time is 10-20 min.
采用上述进一步方案的有益效果在于：通过加入温度为80-100℃的水，使反应后的玉米皮纤维素和半纤维素再生，通过后续工序过滤出来，减少玉米皮小分子多糖的损失。The beneficial effect of adopting the above further scheme is that by adding water at a temperature of 80-100° C., the corn husk cellulose and hemicellulose after the reaction are regenerated and filtered out through subsequent processes, thereby reducing the loss of corn husk small molecular polysaccharides.
进一步，步骤(3)中所述静置时间为10-14h；所述离心速率为5000-12000rmp，离心时间为5-15min。Furthermore, the standing time in step (3) is 10-14 hours; the centrifugal speed is 5000-12000 rpm, and the centrifugal time is 5-15 minutes.
更进一步，步骤(3)中所述所述冷冻干燥温度为-45～-55℃，冷冻干燥时间为12-36hFurthermore, the freeze drying temperature in step (3) is -45 to -55°C, and the freeze drying time is 12 to 36 hours.
采用上述进一步方案的有益效果在于：采用上述工艺对小分子玉米皮多糖进行冷冻干燥，可以避免小分子多糖因黏度特性而粘黏，有利于形成小分子多糖粉末，便于使用与保存，例如测试和复溶等等。The beneficial effect of adopting the above further scheme is that freeze-drying the small molecule corn bran polysaccharide using the above process can avoid the small molecule polysaccharide from sticking due to its viscosity characteristics, which is conducive to the formation of small molecule polysaccharide powder, which is convenient for use and storage, such as testing and re-dissolution.
进一步，步骤(4)中所述分级醇沉的操作为：将玉米皮粗多糖固体粉末进行复溶，然后5000-12000r/min高速离心5-10min分离出水不溶多糖；然后对上清液进行分级醇沉处理，得到的水不溶多糖和各级醇沉多糖样品均进行冷冻干燥，得到小分子玉米皮多糖。Furthermore, the operation of the graded alcohol precipitation in step (4) is as follows: the corn husk crude polysaccharide solid powder is redissolved, and then the water-insoluble polysaccharide is separated by high-speed centrifugation at 5000-12000 r/min for 5-10 min; then the supernatant is subjected to graded alcohol precipitation treatment, and the obtained water-insoluble polysaccharide and each grade of alcohol precipitation polysaccharide samples are freeze-dried to obtain small molecule corn husk polysaccharide.
更进一步，各级醇沉操作为：1)取上清液经旋蒸浓缩，加入无水乙醇，使溶液中乙醇的体积浓度达到40％，在1-4℃下存放10-14h充分醇沉，5000-12000r/min离心5-10min得到40％醇沉多糖固体沉淀，-45～-55℃冷冻干燥12-36h后的固体粉末即为40％醇沉多糖；Furthermore, the alcohol precipitation operation at each level is as follows: 1) taking the supernatant and concentrating it by rotary evaporation, adding anhydrous ethanol to make the volume concentration of ethanol in the solution reach 40%, storing it at 1-4°C for 10-14h to fully precipitate it with alcohol, centrifuging it at 5000-12000r/min for 5-10min to obtain a 40% alcohol-precipitated polysaccharide solid precipitate, and freeze-drying it at -45 to -55°C for 12-36h to obtain a solid powder, which is the 40% alcohol-precipitated polysaccharide;
2)继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到60％，在1-4℃下存放10-14h充分醇沉，5000-12000r/min离心5-10min得到60％醇沉多糖固体沉淀，-45～-50℃冷冻干燥12-36h后的固体粉末即为60％醇沉多糖；2) Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 60%, store at 1-4°C for 10-14h to fully precipitate with alcohol, centrifuge at 5000-12000r/min for 5-10min to obtain a 60% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -45--50°C for 12-36h to obtain a solid powder, which is the 60% alcohol-precipitated polysaccharide;
3)继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到80％，在1-4℃下存放10-14h进行充分醇沉，5000-12000r/min离心
5-10min得到80％醇沉多糖固体沉淀，-45～-50℃冷冻干燥12-36h后的固体粉末即为80％醇沉多糖。3) Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 80%, store at 1-4°C for 10-14h for full alcohol precipitation, and centrifuge at 5000-12000r/min 80% alcohol precipitated polysaccharide solid precipitate is obtained in 5-10 minutes, and the solid powder after freeze-drying at -45 to -50°C for 12 to 36 hours is 80% alcohol precipitated polysaccharide.
采用上述进一步方案的有益效果在于：通过乙醇分级醇沉可以纯化小分子玉米皮多糖的纯度，提纯方法简单，小分子玉米皮多糖的纯度提高。The beneficial effect of adopting the above further scheme is that the purity of small molecule corn bran polysaccharide can be purified by ethanol graded alcohol precipitation, the purification method is simple, and the purity of small molecule corn bran polysaccharide is improved.
本发明的有益效果在于：(1)本发明的技术方案使用酸性低共熔溶剂提取的玉米皮多糖分子量小，工艺简单；The beneficial effects of the present invention are as follows: (1) the molecular weight of corn bran polysaccharide extracted by the technical solution of the present invention using an acidic low eutectic solvent is small and the process is simple;
(2)优选使用乳酸/氯化胆碱低共熔溶剂提取到的玉米皮粗多糖的提取率高，且乳酸与氯化胆碱的摩尔比为10:1，降低了工艺成本。(2) The extraction rate of corn husk crude polysaccharides extracted using the lactic acid/choline chloride low eutectic solvent is high, and the molar ratio of lactic acid to choline chloride is 10:1, which reduces the process cost.
图1为本发明实施例1中HPGPC法测定La/Cc多糖的相对分子质量分布曲线。FIG. 1 is a relative molecular mass distribution curve of La/Cc polysaccharide determined by HPGPC method in Example 1 of the present invention.
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
在本发明实施例中酸性的低共熔溶剂均采用以下方法制备In the embodiments of the present invention, the acidic deep eutectic solvents are prepared by the following method
将氯化胆碱放在鼓风干燥箱中80℃干燥24h，然后按1：2-10的摩尔比称取干燥氯化胆碱和酸，放到具塞锥形瓶中，在80℃的油浴锅中充分搅拌混合1h，直至液体变得透明均一，得到酸性低共熔溶剂。Choline chloride was dried in a forced air drying oven at 80°C for 24 hours, and then the dried choline chloride and acid were weighed in a molar ratio of 1:2-10, put into a stoppered conical flask, and fully stirred and mixed in an oil bath at 80°C for 1 hour until the liquid became transparent and uniform, thereby obtaining an acidic low eutectic solvent.
实施例1Example 1
一种小分子玉米皮多糖的提取方法A method for extracting small molecule corn bran polysaccharide
(1)取玉米皮进行粉碎，然后过40目筛；
(1) taking corn husks and crushing them, and then passing them through a 40-mesh sieve;
(2)使用酸性低共熔溶剂对1g过筛的玉米皮进行120℃加热浸提1h，结束后加入低共熔溶剂5倍体积的80℃水搅拌15min后，过滤分离；酸性低共熔溶剂为乳酸/氯化胆碱，其中酸与氯化胆碱的摩尔比为10：1；(2) using an acidic low eutectic solvent to heat and extract 1 g of sieved corn husk at 120° C. for 1 h, adding 80° C. water (5 times the volume of the low eutectic solvent) and stirring for 15 min, and filtering and separating; the acidic low eutectic solvent is lactic acid/choline chloride, wherein the molar ratio of acid to choline chloride is 10:1;
(3)将过滤液0.1MPa、60℃旋蒸去除水分子至体积不再变化，得到多糖低共熔溶剂浓缩液，向浓缩液中加入浓缩液4倍体积的无水乙醇，4℃下存放静置12h，8000rmp离心分离5min，经无水乙醇洗涤，-50℃冷冻干燥24h得到玉米皮粗多糖；(3) removing water molecules from the filtrate at 0.1 MPa and 60° C. until the volume no longer changes, thereby obtaining a polysaccharide low eutectic solvent concentrate; adding 4 times the volume of anhydrous ethanol to the concentrate; storing the concentrate at 4° C. for 12 h; centrifuging at 8000 rpm for 5 min; washing with anhydrous ethanol; and freeze-drying at -50° C. for 24 h to obtain corn husk crude polysaccharide;
计算粗多糖得率，计算公式见(1)：
Calculate the crude polysaccharide yield using the formula (1):
Calculate the crude polysaccharide yield using the formula (1):
M2——玉米皮粗多糖的质量，g。M 2 ——Mass of corn husk crude polysaccharide, g.
(4)将玉米皮粗多糖固体粉末进行复溶，然后8000r/min高速离心5-min分离出水不溶多糖；然后取上清液经旋蒸浓缩，加入无水乙醇，使溶液中乙醇的体积浓度达到40％，在4℃下存放12h充分醇沉，8000r/min离心5min得到40％醇沉多糖固体沉淀，-50℃冷冻干燥24h后的固体粉末即为40％醇沉多糖；(4) re-dissolving the corn husk crude polysaccharide solid powder, and then centrifuging at 8000 r/min for 5 min to separate the water-insoluble polysaccharide; then taking the supernatant and concentrating it by rotary evaporation, adding anhydrous ethanol to make the volume concentration of ethanol in the solution reach 40%, storing it at 4° C. for 12 h to fully precipitate it with alcohol, centrifuging it at 8000 r/min for 5 min to obtain a 40% alcohol-precipitated polysaccharide solid precipitate, and freeze-drying it at -50° C. for 24 h to obtain a solid powder that is 40% alcohol-precipitated polysaccharide;
继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到60％，在4℃下存放12h充分醇沉，8000r/min离心5min得到60％醇沉多糖固体沉淀，-50℃冷冻干燥24h后的固体粉末即为60％醇沉多糖；Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 60%, store at 4°C for 12 hours to fully precipitate with alcohol, centrifuge at 8000r/min for 5 minutes to obtain a 60% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -50°C for 24 hours to obtain a solid powder that is a 60% alcohol-precipitated polysaccharide;
继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到80％，在4℃下存放12h进行充分醇沉，8000r/min离心5min得到80％醇沉多糖固体沉淀，-50℃冷冻干燥24h后的固体粉末即为80％醇沉多糖。Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 80%, store at 4°C for 12 hours for sufficient alcohol precipitation, centrifuge at 8000r/min for 5 minutes to obtain 80% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -50°C for 24 hours to obtain the solid powder which is 80% alcohol-precipitated polysaccharide.
(5)总多糖纯度和总多糖得率(5) Total polysaccharide purity and total polysaccharide yield
采用苯酚-硫酸法测定粗多糖和各分级醇沉多糖的总多糖纯度，并计算得率。
The phenol-sulfuric acid method was used to determine the total polysaccharide purity of crude polysaccharide and each graded alcohol-precipitated polysaccharide, and the yield was calculated.
1)溶液的配制1) Solution preparation
配制100μg/mL葡萄糖标准溶液：准确称取葡萄糖标准品0.25g，用去离子水溶解于250mL的容量瓶中，定容备用。Prepare 100 μg/mL glucose standard solution: accurately weigh 0.25 g of glucose standard, dissolve it in a 250 mL volumetric flask with deionized water, and make up to volume for later use.
配制6％苯酚溶液：准确称取重蒸苯酚6.00g，去离子水溶解定容于100mL的容量瓶中，现用现配。Prepare 6% phenol solution: Accurately weigh 6.00 g of redistilled phenol, dissolve it in deionized water in a 100 mL volumetric flask, and prepare it before use.
2)总多糖的测定与计算2) Determination and calculation of total polysaccharides
准确吸取0.2mL、0.4mL、0.6mL、0.8mL、1.0mL的葡萄糖标准液到试管中(设置3支平行管)，用去离子水补足至2mL。加入1mL6％的苯酚溶液，摇匀后缓慢加入5mL浓硫酸，沸水浴15min后迅速冷却，测其波长490nm处的吸光度值，以测得490nm处的吸光度值为纵坐标，对应的葡萄糖质量为横坐标，绘制葡萄糖标准曲线。Accurately pipette 0.2mL, 0.4mL, 0.6mL, 0.8mL, and 1.0mL of glucose standard solution into test tubes (set up 3 parallel tubes), and fill to 2mL with deionized water. Add 1mL of 6% phenol solution, shake well, and slowly add 5mL of concentrated sulfuric acid, boil in a water bath for 15min, and then cool rapidly. Measure the absorbance at a wavelength of 490nm, and draw a glucose standard curve with the absorbance at 490nm measured as the ordinate and the corresponding glucose mass as the abscissa.
取适量待测样品测定总多糖纯度，按上述步骤操作，测得其490nm下吸光度值，进而计算不同待测多糖样品中总多糖纯度，计算公式见式(2)。计算三种DESs预处理玉米皮中总多糖分别占玉米皮干重的百分数，即总多糖得率。粗多糖中总多糖得率计算公式见式(3)，各级醇沉多糖中总多糖得率公式见式(4)。
Take an appropriate amount of the sample to be tested to determine the total polysaccharide purity, operate according to the above steps, measure the absorbance value at 490nm, and then calculate the total polysaccharide purity in different polysaccharide samples to be tested. The calculation formula is shown in formula (2). Calculate the percentage of total polysaccharides in corn husks pretreated with three DESs to the dry weight of corn husks, that is, the total polysaccharide yield. The calculation formula for the total polysaccharide yield in crude polysaccharides is shown in formula (3), and the formula for the total polysaccharide yield in each level of alcohol precipitation polysaccharides is shown in formula (4).
Take an appropriate amount of the sample to be tested to determine the total polysaccharide purity, operate according to the above steps, measure the absorbance value at 490nm, and then calculate the total polysaccharide purity in different polysaccharide samples to be tested. The calculation formula is shown in formula (2). Calculate the percentage of total polysaccharides in corn husks pretreated with three DESs to the dry weight of corn husks, that is, the total polysaccharide yield. The calculation formula for the total polysaccharide yield in crude polysaccharides is shown in formula (3), and the formula for the total polysaccharide yield in each level of alcohol precipitation polysaccharides is shown in formula (4).
式中：m2——由标曲查得样品中总多糖质量，g；Where: m 2 ——the total polysaccharide mass in the sample obtained by standard curve, g;
M3——待测样品质量，g。
M 3 ——mass of the sample to be tested, g.
M 3 ——mass of the sample to be tested, g.
式中：M1——玉米皮的质量，g；Where: M 1 ——mass of corn husk, g;
M2——玉米皮粗多糖的质量，g；M 2 ——Mass of corn husk crude polysaccharide, g;
ω7——玉米皮粗多糖中总多糖含量，％；ω 7 ——Total polysaccharide content in corn husk crude polysaccharide, %;
ω8
——玉米皮粗多糖中各级醇沉多糖含量，％； ω8 ——Content of alcohol-precipitated polysaccharides at various levels in corn husk crude polysaccharides, %;
ωi——各级醇沉多糖中总多糖含量，％。ω i ——Total polysaccharide content in each level of alcohol precipitated polysaccharides, %.
结果分析：Result analysis:
经过对多糖分析，多糖的提取率为32％。After polysaccharide analysis, the extraction rate of polysaccharide was 32%.
经过分级醇沉纯化后进行多糖纯度和得率分析，如表1所示。After purification by graded alcohol precipitation, the purity and yield of the polysaccharide were analyzed, as shown in Table 1.
表1粗多糖及分级醇沉
Table 1 Crude polysaccharides and graded alcohol precipitation
Table 1 Crude polysaccharides and graded alcohol precipitation
计算总多糖得率差4％到5％，明面上是水不溶多糖中没测，实际还有80％以上的醇沉多糖，在计算百分比中并没有算此部分多糖，因此导致粗多糖与醇沉总合存在差别。The calculated total polysaccharide yield differs by 4% to 5%. Apparently, this is due to the water-insoluble polysaccharides not being measured, but in fact there are more than 80% of alcohol-precipitated polysaccharides, which are not included in the calculated percentage. This leads to a difference between the crude polysaccharides and the total alcohol-precipitated polysaccharides.
对80％醇沉多糖进行凝胶色谱分析，分析结果如下表2和图1，均为小分子多糖。The 80% alcohol precipitated polysaccharides were subjected to gel chromatography analysis. The analysis results are shown in Table 2 and Figure 1, and all of them are small molecular weight polysaccharides.
表2 80％醇沉多糖分子量
Table 2 Molecular weight of polysaccharide precipitated by 80% alcohol
Table 2 Molecular weight of polysaccharide precipitated by 80% alcohol
对比例1Comparative Example 1
参考卢菲的《玉米皮多糖制备方法及结构研究》，采用水提法提取玉米皮多糖，料水比1：30，提取温度100℃，提取2次，每次2h。在此条件下，100g玉米皮可制备得到6.94g多糖，多糖纯度为88.18％。Referring to Lu Fei's "Study on Preparation Method and Structure of Corn Bran Polysaccharide", corn bran polysaccharide was extracted by water extraction, with a material-water ratio of 1:30, an extraction temperature of 100°C, and 2 extractions, each for 2 hours. Under these conditions, 6.94g of polysaccharide can be prepared from 100g of corn bran, with a polysaccharide purity of 88.18%.
实施例2Example 2
一种小分子玉米皮多糖的提取方法
A method for extracting small molecule corn bran polysaccharide
(1)取玉米皮进行粉碎，然后过60目筛；(1) Grind corn husks and pass through a 60-mesh sieve;
(2)使用酸性低共熔溶剂对过筛的玉米皮进行140℃加热浸提0.5h，结束后加入低共熔溶剂8倍体积的90℃水搅拌10min后，过滤分离；酸性低共熔溶剂为甲酸/氯化胆碱，其中酸与氯化胆碱的摩尔比为2：1；(2) using an acidic low eutectic solvent to heat and extract the sieved corn husk at 140° C. for 0.5 h, adding 8 times the volume of the low eutectic solvent of 90° C. water, stirring for 10 min, and filtering and separating; the acidic low eutectic solvent is formic acid/choline chloride, wherein the molar ratio of acid to choline chloride is 2:1;
(3)将过滤液0.1MPa、60℃旋蒸去除水分子至体积不再变化，得到多糖低共熔溶剂浓缩液，向浓缩液中加入浓缩液5倍体积的无水乙醇，1℃下存放静置10h，5000rmp离心分离15min，经无水乙醇洗涤，-45℃冷冻干燥36h得到玉米皮粗多糖；(3) removing water molecules from the filtrate at 0.1 MPa and 60° C. until the volume no longer changes, thereby obtaining a polysaccharide low eutectic solvent concentrate; adding 5 times the volume of anhydrous ethanol to the concentrate; storing the concentrate at 1° C. for 10 h; centrifuging at 5000 rpm for 15 min; washing with anhydrous ethanol; and freeze-drying at -45° C. for 36 h to obtain corn husk crude polysaccharide;
(4)将玉米皮粗多糖固体粉末进行复溶，然后5000r/min高速离心10min分离出水不溶多糖；然后取上清液经旋蒸浓缩，加入无水乙醇，使溶液中乙醇的体积浓度达到40％，在1℃下存放10h充分醇沉，12000r/min离心5min得到40％醇沉多糖固体沉淀，-45℃冷冻干燥36h后的固体粉末即为40％醇沉多糖；(4) The corn husk crude polysaccharide solid powder is redissolved, and then centrifuged at 5000 r/min for 10 min to separate the water-insoluble polysaccharide; then the supernatant is concentrated by rotary evaporation, anhydrous ethanol is added to make the volume concentration of ethanol in the solution reach 40%, and the solution is stored at 1° C. for 10 h to fully precipitate with alcohol, and centrifuged at 12000 r/min for 5 min to obtain a 40% alcohol-precipitated polysaccharide solid precipitate, and the solid powder after freeze-drying at -45° C. for 36 h is the 40% alcohol-precipitated polysaccharide;
继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到60％，在1℃下存放10h充分醇沉，12000r/min离心5min得到60％醇沉多糖固体沉淀，-45℃冷冻干燥36h后的固体粉末即为60％醇沉多糖；Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 60%, store at 1°C for 10 hours to fully precipitate with alcohol, centrifuge at 12000r/min for 5 minutes to obtain a 60% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -45°C for 36 hours to obtain a solid powder, which is a 60% alcohol-precipitated polysaccharide;
继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到80％，在1℃下存放10h进行充分醇沉，12000r/min离心5min得到80％醇沉多糖固体沉淀，-45℃冷冻干燥36h后的固体粉末即为80％醇沉多糖。Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 80%, store at 1°C for 10 hours for sufficient alcohol precipitation, centrifuge at 12000r/min for 5 minutes to obtain 80% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -45°C for 36 hours to obtain the solid powder which is 80% alcohol-precipitated polysaccharide.
实施例3Example 3
一种小分子玉米皮多糖的提取方法A method for extracting small molecule corn bran polysaccharide
(1)取玉米皮进行粉碎，然后过60目筛；(1) Grind corn husks and pass through a 60-mesh sieve;
(2)使用酸性低共熔溶剂对过筛的玉米皮进行100℃加热浸提3h，结束后加入低共熔溶剂3倍体积的100℃水搅拌20min后，过滤分离；酸性低共熔溶剂为草酸/氯化胆碱，其中酸与氯化胆碱的摩尔比为8：1；
(2) using an acidic low eutectic solvent to heat and extract the sieved corn husk at 100° C. for 3 h, adding 100° C. water (3 times the volume of the low eutectic solvent) and stirring for 20 min, and filtering and separating; the acidic low eutectic solvent is oxalic acid/choline chloride, wherein the molar ratio of acid to choline chloride is 8:1;
(3)将过滤液0.1MPa、60℃旋蒸去除水分子至体积不再变化，得到多糖低共熔溶剂浓缩液，向浓缩液中加入浓缩液4.5倍体积的无水乙醇，4℃下存放静置14h，12000rmp离心分离5min，经无水乙醇洗涤，-55℃冷冻干燥12h得到玉米皮粗多糖；(3) removing water molecules from the filtrate at 0.1 MPa and 60° C. until the volume no longer changes, thereby obtaining a polysaccharide low eutectic solvent concentrate, adding 4.5 times the volume of anhydrous ethanol to the concentrate, storing at 4° C. for 14 h, centrifuging at 12,000 rpm for 5 min, washing with anhydrous ethanol, and freeze-drying at -55° C. for 12 h to obtain corn husk crude polysaccharide;
(4)将玉米皮粗多糖固体粉末进行复溶，然后12000r/min高速离心5min分离出水不溶多糖；然后取上清液经旋蒸浓缩，加入无水乙醇，使溶液中乙醇的体积浓度达到40％，在4℃下存放14h充分醇沉，5000r/min离心10min得到40％醇沉多糖固体沉淀，-55℃冷冻干燥12h后的固体粉末即为40％醇沉多糖；(4) The corn husk crude polysaccharide solid powder is redissolved, and then centrifuged at 12000 r/min for 5 min to separate the water-insoluble polysaccharide; then the supernatant is concentrated by rotary evaporation, anhydrous ethanol is added to make the volume concentration of ethanol in the solution reach 40%, and the solution is stored at 4° C. for 14 h for full alcohol precipitation, and centrifuged at 5000 r/min for 10 min to obtain a 40% alcohol-precipitated polysaccharide solid precipitate, and the solid powder after freeze-drying at -55° C. for 12 h is the 40% alcohol-precipitated polysaccharide;
继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到60％，在4℃下存放14h充分醇沉，5000r/min离心10min得到60％醇沉多糖固体沉淀，-50℃冷冻干燥12h后的固体粉末即为60％醇沉多糖；Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 60%, store at 4°C for 14 hours to fully precipitate with alcohol, centrifuge at 5000r/min for 10 minutes to obtain a 60% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -50°C for 12 hours to obtain a solid powder, which is the 60% alcohol-precipitated polysaccharide;
继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到80％，在4℃下存放14h进行充分醇沉，5000r/min离心10min得到80％醇沉多糖固体沉淀，-50℃冷冻干燥12h后的固体粉末即为80％醇沉多糖。Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 80%, store at 4°C for 14 hours for sufficient alcohol precipitation, centrifuge at 5000r/min for 10 minutes to obtain 80% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -50°C for 12 hours to obtain the solid powder, which is 80% alcohol-precipitated polysaccharide.
实施例4Example 4
一种小分子玉米皮多糖的提取方法A method for extracting small molecule corn bran polysaccharide
(1)取玉米皮进行粉碎，然后过40目筛；(1) taking corn husks, crushing them, and then passing them through a 40-mesh sieve;
(2)使用酸性低共熔溶剂对过筛的玉米皮进行130℃加热浸提2h，结束后加入低共熔溶剂5倍体积的95℃水搅拌12min后，过滤分离；酸性低共熔溶剂为乳酸/氯化胆碱，其中酸与氯化胆碱的摩尔比为5：1；(2) using an acidic low eutectic solvent to heat and extract the sieved corn husk at 130° C. for 2 h, adding 5 times the volume of the low eutectic solvent of 95° C. water, stirring for 12 min, and filtering and separating; the acidic low eutectic solvent is lactic acid/choline chloride, wherein the molar ratio of the acid to the choline chloride is 5:1;
(3)将过滤液0.1MPa、60℃旋蒸去除水分子至体积不再变化，得到多糖低共熔溶剂浓缩液，向浓缩液中加入浓缩液4倍体积的无水乙醇，3℃下存放静置11h，7000rmp离心分离12min，经无水乙醇洗涤，-50℃冷冻干燥18h得到玉米皮粗多糖；
(3) removing water molecules from the filtrate at 0.1 MPa and 60° C. until the volume no longer changes, thereby obtaining a polysaccharide low eutectic solvent concentrate, adding 4 times the volume of anhydrous ethanol to the concentrate, storing at 3° C. for 11 h, centrifuging at 7000 rpm for 12 min, washing with anhydrous ethanol, and freeze-drying at -50° C. for 18 h to obtain corn husk crude polysaccharide;
(4)将玉米皮粗多糖固体粉末进行复溶，然后9000r/min高速离心7min分离出水不溶多糖；然后取上清液经旋蒸浓缩，加入无水乙醇，使溶液中乙醇的体积浓度达到40％，在3℃下存放12h充分醇沉，8000r/min离心10min得到40％醇沉多糖固体沉淀，-45℃冷冻干燥18h后的固体粉末即为40％醇沉多糖；(4) The corn husk crude polysaccharide solid powder is redissolved, and then centrifuged at 9000 r/min for 7 minutes to separate the water-insoluble polysaccharide; then the supernatant is concentrated by rotary evaporation, anhydrous ethanol is added to make the volume concentration of ethanol in the solution reach 40%, and the solution is stored at 3° C. for 12 hours for full alcohol precipitation, and centrifuged at 8000 r/min for 10 minutes to obtain a 40% alcohol-precipitated polysaccharide solid precipitate, and the solid powder after freeze-drying at -45° C. for 18 hours is the 40% alcohol-precipitated polysaccharide;
继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到60％，在2℃下存放11h充分醇沉，11000r/min离心6min得到60％醇沉多糖固体沉淀，-47℃冷冻干燥22h后的固体粉末即为60％醇沉多糖；Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 60%, store at 2°C for 11 hours to fully precipitate with alcohol, centrifuge at 11000r/min for 6 minutes to obtain a 60% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -47°C for 22 hours to obtain a solid powder, which is a 60% alcohol-precipitated polysaccharide;
继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到80％，在1℃下存放13h进行充分醇沉，7000r/min离心8min得到80％醇沉多糖固体沉淀，-48℃冷冻干燥30h后的固体粉末即为80％醇沉多糖。Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 80%, store at 1°C for 13 hours for sufficient alcohol precipitation, centrifuge at 7000r/min for 8 minutes to obtain 80% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -48°C for 30 hours to obtain the solid powder, which is 80% alcohol-precipitated polysaccharide.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.
Claims (9)
- 一种小分子玉米皮多糖的提取方法，其特征在于，包括以下步骤：A method for extracting small molecule corn bran polysaccharides, characterized in that it comprises the following steps:(1)取玉米皮进行粉碎，然后过筛；(1) taking corn husks, crushing them, and then sieving them;(2)使用酸性低共熔溶剂对过筛的玉米皮进行加热浸提，结束后加入低共熔溶剂3-8倍体积的水搅拌分散均匀之后，过滤分离；(2) using an acidic deep eutectic solvent to heat and extract the sieved corn husk, adding 3-8 times the volume of the deep eutectic solvent of water after stirring and dispersing evenly, and filtering and separating;(3)将过滤液旋蒸去除水分子得到多糖低共熔溶剂浓缩液，向浓缩液中加入浓缩液4-5倍体积的无水乙醇，1-4℃下存放静置，离心分离，经无水乙醇洗涤，冷冻干燥得到玉米皮粗多糖；(3) removing water molecules from the filtrate by rotary evaporation to obtain a polysaccharide deep eutectic solvent concentrate, adding 4-5 times the volume of anhydrous ethanol to the concentrate, storing at 1-4° C., centrifuging, washing with anhydrous ethanol, and freeze-drying to obtain corn husk crude polysaccharide;(4)将玉米皮粗多糖经过乙醇分级醇沉纯化得到小分子玉米皮多糖。(4) The crude corn husk polysaccharide is purified by ethanol graded precipitation to obtain small molecular weight corn husk polysaccharide.
- 根据权利要求1所述一种小分子玉米皮多糖的提取方法，其特征在于，步骤(1)中所述过筛粒度为40-60目。The method for extracting small molecule corn bran polysaccharides according to claim 1 is characterized in that the sieving particle size in step (1) is 40-60 mesh.
- 根据权利要求1所述一种小分子玉米皮多糖的提取方法，其特征在于，步骤(2)中所述酸性低共熔溶剂为甲酸/氯化胆碱、草酸/氯化胆碱、甲酸/乙酸/氯化胆碱、乳酸/氯化胆碱中的任一种。The method for extracting small molecule corn bran polysaccharides according to claim 1 is characterized in that the acidic low eutectic solvent in step (2) is any one of formic acid/choline chloride, oxalic acid/choline chloride, formic acid/acetic acid/choline chloride, and lactic acid/choline chloride.
- 根据权利要求3所述一种小分子玉米皮多糖的提取方法，其特征在于，所述酸性低共熔溶剂中酸与氯化胆碱的摩尔比为(2-10)：1。According to claim 3, a method for extracting small molecule corn bran polysaccharides is characterized in that the molar ratio of acid to choline chloride in the acidic low eutectic solvent is (2-10):1.
- 根据权利要求1所述一种小分子玉米皮多糖的提取方法，其特征在于，步骤(2)中所述加热浸提的温度为100-140℃，加热时间为0.5-3h；The method for extracting small molecule corn bran polysaccharides according to claim 1, characterized in that the temperature of the heating extraction in step (2) is 100-140° C. and the heating time is 0.5-3 h;所述加入水的温度为80-100℃，搅拌速率为60-300rmp，搅拌时间为10-20min。The temperature of adding water is 80-100° C., the stirring rate is 60-300 rpm, and the stirring time is 10-20 min.
- 根据权利要求1所述一种小分子玉米皮多糖的提取方法，其特征在于，步骤(3)中所述静置时间为10-14h；所述离心速率为5000-12000rmp，离心时间为5-15min。According to the method for extracting small molecule corn bran polysaccharides according to claim 1, it is characterized in that the standing time in step (3) is 10-14h; the centrifugal rate is 5000-12000rmp, and the centrifugal time is 5-15min.
- 根据权利要求1所述一种小分子玉米皮多糖的提取方法，其特征在于，步骤(3)中所述所述冷冻干燥温度为-45～-55℃，冷冻干燥时间为12-36h The method for extracting small molecule corn bran polysaccharides according to claim 1, characterized in that the freeze-drying temperature in step (3) is -45 to -55°C and the freeze-drying time is 12 to 36 hours.
- 根据权利要求1所述一种小分子玉米皮多糖的提取方法，其特征在于，步骤(4)中所述分级醇沉的操作为：将玉米皮粗多糖固体粉末进行复溶，然后5000-12000r/min高速离心5-10min分离出水不溶多糖；然后对上清液进行分级醇沉处理，得到的水不溶多糖和各级醇沉多糖样品均进行冷冻干燥，得到小分子玉米皮多糖。According to claim 1, a method for extracting small molecule corn bran polysaccharides is characterized in that the operation of the graded alcohol precipitation in step (4) is: the corn bran crude polysaccharide solid powder is redissolved, and then the water-insoluble polysaccharide is separated by high-speed centrifugation at 5000-12000r/min for 5-10min; then the supernatant is subjected to graded alcohol precipitation treatment, and the obtained water-insoluble polysaccharides and the various levels of alcohol precipitation polysaccharide samples are freeze-dried to obtain small molecule corn bran polysaccharides.
- 根据权利要求8所述一种小分子玉米皮多糖的提取方法，其特征在于，各级醇沉操作为：1)取上清液经旋蒸浓缩，加入无水乙醇，使溶液中乙醇的体积浓度达到40％，在1-4℃下存放10-14h充分醇沉，5000-12000r/min离心5-10min得到40％醇沉多糖固体沉淀，-45～-55℃冷冻干燥12-36h后的固体粉末即为40％醇沉多糖；According to claim 8, a method for extracting small molecule corn bran polysaccharides, characterized in that the alcohol precipitation operations at each level are as follows: 1) taking the supernatant and concentrating it by rotary evaporation, adding anhydrous ethanol to make the volume concentration of ethanol in the solution reach 40%, storing it at 1-4°C for 10-14h to fully precipitate it with alcohol, centrifuging it at 5000-12000r/min for 5-10min to obtain a 40% alcohol-precipitated polysaccharide solid precipitate, and freeze-drying it at -45 to -55°C for 12-36h to obtain a solid powder that is a 40% alcohol-precipitated polysaccharide;2)继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到60％，在1-4℃下存放10-14h充分醇沉，5000-12000r/min离心5-10min得到60％醇沉多糖固体沉淀，-45～-50℃冷冻干燥12-36h后的固体粉末即为60％醇沉多糖；2) Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 60%, store at 1-4°C for 10-14h to fully precipitate with alcohol, centrifuge at 5000-12000r/min for 5-10min to obtain a 60% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -45--50°C for 12-36h to obtain a solid powder, which is the 60% alcohol-precipitated polysaccharide;3)继续回收上清液，旋蒸浓缩后加入无水乙醇，使溶液中乙醇的体积浓度达到80％，在1-4℃下存放10-14h进行充分醇沉，5000-12000r/min离心5-10min得到80％醇沉多糖固体沉淀，-45～-50℃冷冻干燥12-36h后的固体粉末即为80％醇沉多糖。 3) Continue to recover the supernatant, add anhydrous ethanol after rotary evaporation concentration to make the volume concentration of ethanol in the solution reach 80%, store at 1-4°C for 10-14h for sufficient alcohol precipitation, centrifuge at 5000-12000r/min for 5-10min to obtain 80% alcohol-precipitated polysaccharide solid precipitate, and freeze-dry at -45 to -50°C for 12-36h to obtain the solid powder, which is 80% alcohol-precipitated polysaccharide.
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