多糖纯化的意义在于去除小分子，如色素和无机盐。目前，分离方法包括色谱和透析。纯化方法可以根据分子量和中等酸度的标准对多糖进行分类。由于其生物活性和无毒或低毒特性，食品来源的天然多糖在食品、化妆品、生物材料等领域越来越受欢迎[37，38，39，40，41，42]。纯化还可以进行可用、安全和可重复的多糖研究[43]。纯化方法的选择取决于多糖的性质，并受制造工艺的影响。在原料预处理过程中未完全去除的杂质（淀粉、脂质和色素）可能在随后的多糖提取过程中与多糖混合。此外，最近的研究表明，不同的提取条件，如pH和溶剂温度，都会导致不同多糖的渗透[44，45]。如图4所示，Wei等人[40]将多糖的分离分为三个阶段：原料中的多糖，粗提物中的多糖和粗多糖。粗多糖的纯化包括多糖的富集和具有不同结构或构象特征的馏分的分离。

Polysaccharides have been widely accepted as nutraceuticals, which improve the immune function of the body. However, many polysaccharides remain as health products and fail to be developed into drugs. The main reason for this is that the separation and purification of polysaccharides is difficult, and the current technology level does not meet the requirements for this. Generally speaking, polysaccharides are hydrophilic macromolecules. The separation and purification methods of polysaccharides are different from those of small molecules. In addition, different polysaccharides have different properties, so different separation and purification methods must be used. This work requires not only a theoretical knowledge of polysaccharides, but also accumulated working experience in the separation and purification of polysaccharides.

This method mainly takes advantage of the fact that the solubility of different polysaccharides in different concentrations of organic solvents is different. The solubility of polysaccharides with larger molecular weights in ethanol or acetone are less than it is of those with a smaller molecular weight [46]. Therefore, the molecular weight of the product can be controlled by adjusting the concentration of the organic solvent. This is usually done in the following manner:

While stirring, a high concentration of anhydrous ethanol is slowly added to the solution of the polysaccharide mixture to reach a final concentration of 25% ethanol (v/v). After the addition of ethanol, the solution is left for 2 h and then centrifuged to obtain the supernatant and precipitate (which may be referred to as the “first precipitate”). The precipitate is of a high MW polysaccharide grade. While stirring, ethanol is slowly added to the supernatant to reach a final concentration of 35% ethanol (v/v). The solution is left for 2 h and then centrifuged to obtain the supernatant and precipitate (which may be referred to as the “second precipitate”). The second precipitate is also a polysaccharide fraction, but its MW is lower than the first precipitate. The step-down process can be carried out further, depending on circumstances. The key to graded precipitation is to avoid co-precipitation as much as possible. The concentration of the polysaccharide mixture should not be too high, the ethanol should not be added too fast, and the pH of the solution should be near neutral. The lower the concentration of polysaccharide solution, the weaker the co-precipitation effect and the better the purification effect. However, if the polysaccharide concentration is too low, the recovery of the polysaccharide will be reduced and the consumption of ethanol will be greatly increased. Usually, the concentration of polysaccharide in the mixture is adjusted from 0.25% (w/v) to 3% (w/v) before using this method [47,48,49,50]. The concentration grading method is commonly used in the research and development of polysaccharide nutraceuticals because it is much easier than column chromatography.

Column chromatography is the most widely used method for the purification of polysaccharides. Several methods of column chromatography are described, as follows:

Macroporous adsorption resin is used to selectively adsorb organic substances from the solution by physical adsorption so as to achieve separation and purification. Its physical and chemical properties are stable; it is insoluble in acids, bases, and organic solvents, has good selectivity for organic substances, and it is unaffected by the presence of inorganic salts and strong ions, low molecular compounds, and swelling in water and organic solvents by the adsorption of solvents. The adsorption of macroporous resin relies on the van der Waals gravitational force between it and the adsorbed molecules (adsorbent), and works through its huge specific surface for physical adsorption, so that organic compounds can be separated by a certain solvent elution according to the adsorption force and its molecular weight size to achieve different purposes such as separation, purification, de-hybridization, and concentration. Macroporous resin can remove proteins, flavonoids, and pigments from polysaccharide solutions [51,52,53,54].

Cellulose is a common filling material in columns. First, after waiting for swelling, activation is performed using 0.5 mol/L NaOH with 0.5 mol/L HCL solution; the cellulose in the column is equilibrated with NaCL solution, and then the polysaccharide is loaded onto the cellulose column for purification. Afterwards, the cellulose columns are eluted separately using an eluent so that different polysaccharide levels can be continuously eluted [46]. Polysaccharides can be separated according to different molecular weights or acid-based groups. In the elution process, the various polysaccharide fractions undergo several dissolution and precipitation processes in the cellulose column, and can eventually be separated from each other. This method can be called the “graded dissolution method”, which is basically the opposite of the graded precipitation method. Due to the high number of theoretical plates in the cellulose column chromatography, the purity of the eluate is higher [55]. However, the disadvantage of this method is the low flow rate and the long period of time required. The flow rate seems to be too low, especially for highly viscous acidic polysaccharides.

Gel column chromatography is based on the size and shape of polysaccharide molecules, i.e., the molecular sieve principle, to separate polysaccharides. This chromatographic method is widely used for the separation and purification of polysaccharides. In general, the crude polysaccharides obtained are first purified using macroporous resin and cellulose chromatography, and are then further purified using gel column chromatography. Commonly used gels are various types of Sephadex, Sepharose, Bio gels, and later Sephacryl, Superdex, and Superose. The eluents are salt solutions and buffers of various concentrations.