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Auricularia auricula polysaccharides (黑木耳多糖（AAP）因其独特的结构和生理活性而在医学和保健领域得到了广泛的研究。许多种类黑木耳多糖已通过不同的方法提取、分离和纯化，并对其结构进行了分析。黑木耳多糖已被证明对人体有益，包括减缓衰老过程、控制肠道系统和治疗心血管疾病。本文介绍了AAP的提取、分离和纯化黑木耳以及医学和医疗保健领域的研究，都指出了这些方法的缺点和局限性。我们还提出了未来的研究方向黑木耳多糖;必须确认标准化的加工方法，并且商业应用需要官方批准的AAP。最后，对AAP) have been widely studied in the field of medicine and healthcare because of their unique structure and physiological activity. Many species of Auricularia auricula polysaccharides have been extracted, isolated, and purified by different methods, and their structures have been analyzed. Auricularia auricula polysaccharides have been proven to have beneficial effects on the human body, including slowing the aging process, controlling the intestinal system, and treating cardiovascular disorders.的发展给出了乐观的展望。
- Auricularia auricula
- polysaccharide
- extraction technology
1. Introduction简介
Auricularia auricula is an edible mushroom and a traditional medicine in China, and it is also the fourth largest cultivated mushroom species in the world [1]. It is widely valued in Asia and around the world for its nutritional and healing properties. China, in particular, is a major producer of Auricularia auricula, which has been consumed for at least 耳蕈是食用菌，是中国的传统药物，也是世界第四大栽培蘑菇品种[1]。它因其营养和治疗特性而在亚洲和世界各地受到广泛重视。特别是中国是耳廓的主要生产国，耳廓菌已被食用至少4000 years and is used as a common traditional medicine in Asia [2][3].年，在亚洲被用作常见的传统药物[2，3]。
Auricularia auricula is rich in polysaccharides, proteins, fats, vitamins, pigments, and trace elements [4]. Auricularia auricula polysaccharide (耳廓富含多糖、蛋白质、脂肪、维生素、色素和微量元素[4]。耳廓多糖（AAP) is the main active ingredient of Auricularia auricula, accounting for ）是耳廓的主要活性成分，占总含量的60% of the total content [5][6]. In recent years, the research on [5，6]。近年来，医疗保健领域对AAPs in the field of healthcare has intensified, and a lot of chronic disorders have been found to respond quite favorably to its therapeutic effects [7].的研究已经加强，并且发现许多慢性疾病对其治疗效果的反应相当有利[7]。
Many studies have confirmed that polysaccharides from Auricularia auricula can regulate intestinal flora [8], enhance immune regulation [9], slow down the aging process [10][11], resist radiation [12], reduce blood lipids [13], and exterminate viruses [14]. These activities show great promise for the development of nutritional medicines and pharmaceuticals. The health benefits from 许多研究证实，耳廓多糖可以调节肠道菌群[8]，增强免疫调节[9]，减缓衰老过程[10，11]，抵抗辐射[12]，降低血脂[13]，消灭病毒[14]。这些活动对营养药物和药物的发展显示出巨大的希望。AAP go far beyond the other essential nutrients in Auricularia auricula, and are a major direction for the future deep processing of Auricularia auricula.的健康益处远远超过耳廓中的其他必需营养素，是未来耳廓深加工的主要方向。
Because the polysaccharide is a Fungi heteropolysaccharide, the uncertainty of its structure and composition has caused great difficulties for its in-depth study. The biological activity of the samples obtained by different extraction or drying methods was different [15]. Therefore, the study of extraction technology is the first hurdle in the in-depth study of the polysaccharides of Auricularia auricula. To improve the yield of polysaccharides, scientists have developed enzymatic extraction [16], ultrasonic extraction [17], liquid fermentation extraction [18], and other methods in recent years. 由于多糖是一种真菌杂多糖，其结构和组成的不确定性给其深入研究带来了很大的困难。不同提取或干燥方法获得的样品的生物活性不同[15]。因此，提取技术的研究是深入研究耳廓多糖的第一道坎。为了提高多糖的收率，近年来科学家开发了酶法提取[16]、超声提取[17]、液体发酵提取[18]等方法。就目前获得的多糖而言，主要研究了四种类型的多糖（角苣苔（As far as the polysaccharides obtained so far are concerned, four types of polysaccharides (A. corna (ACP), A. auricula (AAP), A. polytricha (APP), and M. murgy (MFP)) have mainly been studied [19]. Finding an extraction method with high reproducibility of the extracted sample structure and the molecular weight is a key research direction for the extraction of ）、耳廓苔（AAP）、多耳苣苔（APP）和默氏多糖（MFP））[19]。寻找对提取样品结构和分子量具有高重现性的提取方法是AAP. Moreover, the abovementioned traditional method of extracting Auricularia auricula polysaccharides leads to a large amount of cooking waste liquid and a large amount of nutrient loss. Recent studies have shown that the melanin in the residual species of the remaining Auricularia auricula has a great potential for resource exploitation, enabling the recultivation of other plant species on the waste and facilitating the preparation of beverages from whey protein isolates [20][21][22]. The reuse of Auricularia auricula residue will also be a future research direction.提取的重点研究方向。而且，上述提取耳廓多糖的传统方法导致大量的烹饪废液和大量的营养损失。最近的研究表明，剩余的耳廓残留物种中的黑色素具有巨大的资源开发潜力，能够在废物上重新培养其他植物物种，并促进从乳清蛋白分离物制备饮料[20，21，22]。耳苣苔残留物的再利用也将是未来的研究方向。
China is currently the world’s largest producer of Auricularia auricula, accounting for more than 中国目前是世界上最大的耳廓生产国，占全球总产量的90% of the total global production. In 以上。2018, the output of Auricularia auricula (dried products) reached 年，耳苋（干货）产量达到67.4,000 tons, with an output value of 37.46 billion yuan and a foreign exchange generation of 6.15 billion yuan. As a characteristic and advantageous agricultural product in China, mushrooms have played an important role in improving agricultural efficiency, increasing farmers’ incomes, and helping to alleviate poverty in the industry [20]. It is also important to note that the production scale of Auricularia auricula cultivation has increased year by year, and the market pressure on Auricularia auricula forest products has also been increasing with the popularization and application of bagging cultivation technology [21]. The cultivation scale of Auricularia auricula is expanding, the production is increasing year by year, and the profit margin of market sales is gradually shrinking. A gradual change from a seller’s market to a buyer’s market has been occurring. The deep processing of Auricularia auricula is one of the future development directions of Auricularia auricula products. Figure 万吨，产值374.6亿元，创汇61.5亿元。作为我国特色优势农产品，蘑菇在提高农业效益、增加农民收入、助力行业扶贫等方面发挥着重要作用[20]。需要注意的是，耳廓栽培的生产规模逐年增加，随着套袋栽培技术的普及应用，耳木林产品的市场压力也在不断增加[21]。耳廓种植规模不断扩大，产量逐年增加，市场销售利润空间逐渐萎缩。从卖方市场到买方市场正在逐步转变。耳廓深加工是耳廓产品未来发展方向之一。图1 shows the change in Auricularia auricula production in China from 显示了2016 to 年至2022, which has been growing steadily since 年中国耳廓产量的变化，自2018, reaching 年以来一直在稳步增长，到2020年达到7,295,900 tons by 2020, an increase of 3.96% year-over-year [22]. By ，295，900吨，同比增长3.96%[22]。到2022, China’s Auricularia auricula production will grow to 年，中国的耳廓产量将增长到7,，723,400 tons, up 1.98% year-over-year [23]. With the continuous updating of cultivation technology and culture methods, further development of Auricularia auricula polysaccharide products and the deep processing of them is the way forward for the Auricularia auricula industry. As shown in Figure ，400吨，同比增长1.98%[23]。随着栽培技术和培养方法的不断更新，耳廓多糖产品的进一步发展及其深加工是耳木耳产业的前进方向。如图2, among all Auricularia auricula deep processing products, Auricularia auricula beverage products have the largest market share at 所示，在所有耳廓深加工产品中，耳廓饮料产品的市场份额最大，为28%, which is much higher than that for other products. The product with the second highest market share is Auricularia auricula nutritional powder, with an ，远高于其他产品。市场份额第二高的产品是耳廓营养粉，市场份额为8% market share followed by Auricularia auricula porridge and ready-to-eat Auricularia auricula, each with a ，其次是耳廓粥和即食耳廓，各占6% market share [22][24][25]. Therefore, in the Chinese market, Auricularia auricula is mainly used in products that help to lower blood lipids and enhance immunity.的市场份额[22，24，25]。因此，在中国市场，耳廓主要用于有助于降低血脂和增强免疫力的产品中。
Figure
图1.
Production and forecast of Auricularia auricula in China from 2016–2022.
Figure
2016-2022年中国耳廓菌产量与预测.
图2.
The proportion of commodity types in the Auricularia auricula market.
The demand for polysaccharides as nutritional and functional components of Auricularia auricula is growing, and the market is expanding [26][27]. Many high-value (耳廓市场的商品类型比例。
作为耳廓多糖的营养和功能成分的需求正在增长，市场正在扩大[26，27]。许多高价值（AAP) derivatives have been developed, and processing technologies have matured. ）衍生物已经开发出来，加工技术已经成熟。虽然它们对健康有很多好处，但它们还没有完全上市。耳廓多糖具有独特的结构，具有广泛的生物学和药理活性。由于其高含量、易于提取、特定结构、轻微的副作用和显着的治疗潜力，它们在世界范围内越来越受欢迎。到目前为止，已经从分布在世界各地的不同黑曲霉物种中提取了许多Although they have many health benefits, they are not yet fully to market. Auricularia auricula polysaccharides have a unique structure with a wide range of biological and pharmacological activities. They are becoming increasingly popular worldwide due to their high content, ease of extraction, specific structure, minor side effects, and significant therapeutic potential. So far, many AAPs have been extracted from different [11]。对不同黑曲霉物种的A. nigra species distributed all over the world [11]. Numerous in-depth studies on the structure and biological and pharmacological activities of AAPs from different 的结构和生物学和药理活性进行了大量深入研究。然而，标准化的加工方法尚未得到确认，A. nigra species have been carried out. However, standardized processing methods have yet to be confirmed, and the commercial applications of AAP need to be officially approved. The aim of this research is to provide useful information and practical recommendations for the large-scale commercial production of AAP and to enable the rapid market entry of promising functional ingredients with economic value for the food industry and health benefits for consumers.AP的商业应用需要官方批准。
To improve the commercial value of Auricularia auricula polysaccharides, the creation of a new grading standard is necessary. Investigating the effects of different molecular weights, different monosaccharide compositions, and different spatial structures on their physiological activities is of paramount importance.
2. Pretreatment of Auricularia auricula and Extraction Technology分离纯化
Pretreatment is the initial ste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p before extracting polysaccharides from fungi. Its major objective is to eliminate undesired components while retaining polysaccharides. Auricularia auricula must be pretreated to obtain pure products. The protein and ash are frequently removed while treating Auricularia auricula. The related pretreatments of Auricularia auricula are provided in Table 1. The yields and physiological activities of polysaccharides obtained from different regions of the fungus using different extraction methods and different drying methods are very different.Table 1. Pretreatment, extraction, and separation of AAPs from different Auricularia auricula.
| Origin | Extraction Method | Time (min) | Solid-Liquid Ratio | Temperature (°C) | Other Conditions | Yield (%) | References |
|---|---|---|---|---|---|---|---|
| Jinlin County, Jilin Province | Ultrasonic Assist | 40 | 1:70 | 70 °C | Particle size of 150–200 mesh | 29.29 ± 1.41% | [28] |
| Qingchuan County, Sichuan Province | MAE | 25 | 1:25 | 95 °C | Microwave power of 860 W, pH 7.0 | 10.52% | [29] |
| Jiaohe County, Jilin Province | Pulsed Electric Field | 1.5 | 1:30 | Room temperature | HIPEF strength at 24 kV/cm, pulse number at 6, pH 8 | 14.79% | [30][31] |
| Jinan | PEG-based ultrasound-assisted | 32.44 | 1:39.27 | 91.948 °C | PEG concentration of 0.30 g/mL | 21.58% | [32] |
| Greater Khingan Mountains, Helongjiang | Neutral protease | / | 1:75 | 50 °C | E/S at 8% | 12.96% | [13] |
| Greater Khingan Mountains, Heilongjiang Province | Ultrasonic Assist Alkali method | 90 | 1:48 | 70 °C | 2.0% of NaOH concentration | 15.53% | [33] |
| Greater Khingan Mountains, Heilongjiang Province | Mannanase, β-dextranase, and cellulase | 60 | 1:80 | 83.17 °C | pH at 2.1 | 26.42% ± 0.87% | [34] |
Auricularia auricula polysaccharides account for 60% of its total content [35]. However, the yield of the current extraction process is not ideal, and improving the polysaccharide yield and enhancing its specific activity is the direction of further research on Auricularia auricula polysaccharides.
As far as the process of extracting polysaccharides is concerned, the raw material of Auricularia auricula is treated in two ways, as shown in Figure 3. The polysaccharides obtained by the two treatments were mainly composed of rhamnose, galactose, glucose, mannose, and xylose, but the molar ratios were different. The polysaccharides obtained from the fermentation broth of Auricularia auricula were mostly extracellular polysaccharides, which have better oxidation resistance, but the yield was lower than that of powdered raw materials [26][36].
Figure 3. Auricularia auricula has two types of handling for raw materials.
Researchers also found that the particle size of Auricularia auricula powder has a great influence on its extraction rate. The smaller the particle size, the higher the extraction rate. The intracellular polysaccharides of Auricularia auricula are largely restricted by the cell wall. The first step in extracting polysaccharides is to destroy the cell structure. This can be done by ultrasound, high temperature, or other methods, and the basic principle is to destroy the cell structure and increase the free constant of Auricularia auricula polysaccharide so as to obtain the target sample.
From the best conditions of these methods, several conclusions can be inferred (except the enzyme method): (1) The smaller the particle size of comminution, the higher the extraction rate of polysaccharides; (2) The higher the extraction temperature and pressure, the higher the extraction rate of polysaccharides; (3) The longer the extraction time, the higher the extraction rate of polysaccharides. The effects of temperature, pressure, and time can be attributed to cell destruction theory. However, the particle size of Auricularia auricula powder is also related to its water absorption, referred to as “water absorption asymmetry of feather cells in polysaccharides”. This effect leads to the different stretching of polysaccharides and cell walls during hot water immersion, so the separation effect can be achieved. According to this principle, it can be inferred that the water absorption strength of Auricularia auricula may be related to the content of polysaccharides in the body. In order to further improve the extraction rate of Auricularia auricula polysaccharides, the cell wall should be destroyed as much as possible, and the particle size should be as large as the polysaccharide molecule.
In addition, according to this research, the cooking liquid obtained during the processing of Auricularia auricula is brown or dark black, and the protein content is high. The polysaccharides extracted from the body wall and cooking liquid of sea cucumber are brown. Therefore, it is suggested that the raw materials of the Auricularia auricula body wall should be pretreated to remove pigments and inorganic salts as much as possible. Otherwise, the obtained coarse AAP is dark brown with a high ash content. In addition, from the perspective of industrial applications, since organic solvents are not allowed to be used in the food and pharmaceutical industries, it is necessary to establish an alternative and effective protein removal method.
3. Separation and Purification
The significance of polysaccharide purification lies in the removal of small molecules, such as pigments and inorganic salts. Currently, separation methods include chromatography and dialysis. Purification methods can classify polysaccharides according to the criteria of molecular weight and moderate acidity. Because of their biological activity and non-toxic or low-toxicity properties, natural polysaccharides of food origin are gaining popularity in food, cosmetics, biomaterials, and other fields [37][38][39][40][41][42]. Purification also enables usable, safe, and reproducible polysaccharide research [43]. The choice of purification method is determined by the nature of the polysaccharide and is influenced by the manufacturing process. Impurities (starch, lipids, and pigments) that are not completely removed during raw material pretreatment may mix with polysaccharides during the subsequent polysaccharide extraction process. Furthermore, recent research has shown that different extraction conditions, such as pH and solvent temperature, can result in the permeation of different polysaccharides [44][45]. As shown in Figure 和溶剂温度，都会导致不同多糖的渗透[44，45]。如图4, 所示，Wei et al. [40] divided the separation of polysaccharides into three stages: polysaccharides in raw material, polysaccharides in crude extract, and crude polysaccharides. The purification of crude polysaccharides includes the enrichment of polysaccharides and the separation of fractions with different structural or conformational characteristics.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。
Figure 4. The purification process of polysaccharides.
The purification process of polysaccharides.
3.1. Concentration Grading Method
2.1. Concentration Grading Method
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][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.3.2. Column Chromatography Method
2.2. Column Chromatography Method
Column chromatography is the most widely used method for the purification of polysaccharides. Several methods of column chromatography are described, as follows:32.2.1. Macroporous Resin
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][51,52,53,54].32.2.2. Cellulose Column Chromatography
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.32.2.3. Gel Column Chromatography
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.4. Physiological Activity and Product Development of Polysaccharides of Auricularia auricula
4.1. Physiological Activity of Auricularia auricula
4.1.1. Regulation of Intestinal Flora
The intestinal flora is essential for maintaining host health by regulating cellular activity and the immune system. According to previous studies, it is associated with leukemia infection [56], small bowel colitis [57], ischemic stroke [58], obesity [59], and a number of other harmful conditions. According to the research, as shown in Figure 5, there are as many as 51 metabolites regulated by Auricularia auricula polysaccharide, which are mainly concentrated in the arginine biosynthesis pathway, followed by the arginine and proline, glycine, serine, and threonine, glycerophospholipids, and sphingolipid metabolic pathways [60]. These pathways can lower total and LDL cholesterol levels and alter the composition of the intestinal flora. The relative abundance levels of Lactobacillus johnsonii, Weissella cibaria, Kosakonia covanii, Enterococcus faecalis, Bifidobacterium animalis, and Bacteroides uniformis are significantly upregulated, while Firmicutes bacteria m10-2 are downregulated. The biological activity of AAP may be related to the regulation of the endogenous metabolism and intestinal flora composition. Zhang et al. [8]. found that A. auricular upregulated the high-abundance SCFA-producing genus Bacteroides and Paraprevotella in a dietary fiber-rich diet, while AAP could better enrich several lower-abundance SCFA-producing bacteria, such as Flavonifractor and Clostridium IV.
Figure 5. Mechanism of Auricularia auricula polysaccharides.
