WO2022067774A1 - 一种海参多糖的制备方法与应用 - Google Patents

一种海参多糖的制备方法与应用 Download PDF

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WO2022067774A1
WO2022067774A1 PCT/CN2020/119685 CN2020119685W WO2022067774A1 WO 2022067774 A1 WO2022067774 A1 WO 2022067774A1 CN 2020119685 W CN2020119685 W CN 2020119685W WO 2022067774 A1 WO2022067774 A1 WO 2022067774A1
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sea cucumber
cucumber polysaccharide
polysaccharide
pure water
ultrafiltration
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PCT/CN2020/119685
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English (en)
French (fr)
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李振国
郑顺亮
李伟
胡杰文
南志远
周剑波
郭予茜
李长江
张凯
夏珂
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牡丹江友搏药业有限责任公司
九芝堂股份有限公司
海南九芝堂药业有限公司
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Priority to PCT/CN2020/119685 priority Critical patent/WO2022067774A1/zh
Publication of WO2022067774A1 publication Critical patent/WO2022067774A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/616—Echinodermata, e.g. starfish, sea cucumbers or sea urchins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof

Definitions

  • the invention relates to a sea cucumber polysaccharide and a preparation method and application thereof.
  • Sea cucumber is a traditional nourishing Chinese medicinal material, and its efficacy is highly recognized, but the processing method and taking method are simple and simple, the absorption effect cannot be guaranteed, and it is easy to cause waste of nutrients.
  • sea cucumber is a traditional health product in my country with high health and nutritional value, and it is also an important research object of marine functional food.
  • sea cucumber polysaccharide has various biological activities such as anti-hyperlipidemia, anti-oxidation, anti-coagulation, anti-tumor, and immunity enhancement.
  • sea cucumber polysaccharide mainly focuses on the body wall of sea cucumber, and the body wall polysaccharide includes two components: sea cucumber chondroitin sulfate (SC-CHS) and sea cucumber fucoidan sulfate (SC-FUC) (Zhang Jian et al., Sea cucumber polysaccharide preparation, Research progress in chemical analysis and biological activity [J], Journal of Food Safety and Quality Inspection, 2018, 9(08):1730-1736).
  • SC-CHS sea cucumber chondroitin sulfate
  • SC-FUC sea cucumber fucoidan sulfate
  • Sea cucumber fucoidan sulfate is one of the main functional components of sea cucumber, belongs to the fucoidan sulfate compound, has a variety of physiological regulation functions, and shows a good application prospect in the development of functional food .
  • SC-FUC Sea cucumber fucoidan sulfate
  • the existing traditional extraction process of sea cucumber polysaccharide can be divided into five categories: acid method, alkaline method, enzymatic method, salt method and hot water extraction method, etc.
  • the basic principle is to change the external environment of protein according to the characteristics of seafood, to separate the polysaccharides. Since the above method will use a large amount of fresh water and acid-base salts in the extraction process, it is a high-energy-consumption industry, and the discharged acid-base salts have a certain ecological impact on the surrounding environment, which is not in line with the green sustainable economic development model, and the extraction process relatively rough.
  • the technical problem to be solved by the present invention is to provide a method for extracting sea cucumber polysaccharide by combining biological enzymolysis technology, salting-out alcohol precipitation method, column chromatography purification and decolorization and membrane separation technology desalination, which can not only reduce energy consumption, but also improve the Extraction rate of sea cucumber polysaccharides.
  • the present invention provides the following technical solutions:
  • a method for preparing sea cucumber polysaccharide comprising the following steps:
  • step 1) the dried sea cucumber is pulverized (pulverized into coarse particles), pure water 8-12 times the weight of the sea cucumber is added, and papain is used for enzymatic hydrolysis.
  • the sea cucumber is crushed into coarse particles, which is conducive to the reaction.
  • the enzymatic hydrolysis conditions are as follows: the amount of papain added is 1%-3% of the weight of the sea cucumber, the pH is 6.0-7.0, the temperature is 50°C-60°C, and the time is 4-6h.
  • the enzymatic hydrolysis conditions are as follows: the amount of papain added is 3% of the weight of the sea cucumber, the pH is 6.5, the temperature is 60°C, and the time is 6h.
  • the centrifugal speed is 4000-15000rpm. More preferably, the centrifugation speed is 15000 rpm.
  • the salting-out alcohol precipitation treatment includes the steps: adding potassium acetate to the supernatant to a final concentration of 0.5M, adding ethanol to a final concentration of 60% (v/v), and centrifuging to obtain Precipitation A; Precipitation A Dissolve with pure water, add potassium acetate to a final concentration of 1.0M, add ethanol to a final concentration of 40% (v/v), centrifuge to obtain precipitate B; dissolve precipitate B with pure water, add potassium acetate to a final concentration of 1.0 M, add ethanol to a final concentration of 40% (v/v), centrifuge to obtain a precipitate C; wash the precipitate C with absolute ethanol, evaporate the ethanol to dryness, and obtain a crude sea cucumber polysaccharide.
  • the concentration of NaCl solution is 0.5-3.0mol/L.
  • step 2) in, the sea cucumber polysaccharide crude product is dissolved with pure water, go on D204 type anion chromatography column (OH - ), successively use 2-3 times of column volumes of deionized water, 2-3 times of column volumes of 0.3- 0.6mol/L NaCl solution, 2-3 column volumes of 0.9-1.2mol/L NaCl solution, 2-3 column volumes of 1.8-2.4mol/L NaCl solution gradient elution, the elution flow rate is 2BV /h, collect the eluate when eluted with 1.8-2.4mol/L NaCl solution. Then wash the column with about 3 mol/L NaCl solution.
  • the column bed volume of pure water and 0.5, 1.0, and 2.0 mol/L NaCl solution are used for gradient elution respectively, and the eluate when eluted with 2.0 mol/L NaCl solution is collected. Then wash the column with about 3 mol/L NaCl solution.
  • step 3 the eluate collected in step 2) is subjected to ultrafiltration desalination treatment on a 5KD hollow fiber column, and the ultrafiltration retentate is collected and concentrated under reduced pressure at 50°C-60°C and vacuum degree ⁇ -0.085MPa, Freeze-drying to obtain sea cucumber polysaccharide.
  • the ultrafiltration conditions are: reflux pressure ⁇ 0.15MPa, when the volume of the ultrafiltration retentate reaches half of the volume before ultrafiltration, start to replenish water, and replenish water to the number of liquid volumes before ultrafiltration; ultrafiltration behavior Continue until the ultrafiltration retentate and Cl - in the permeate are negative.
  • Another object of the present invention is to provide a sea cucumber polysaccharide prepared by any of the above methods.
  • Another object of the present invention is the application of the sea cucumber polysaccharide prepared by any of the above methods in the preparation of anti-hyperlipidemia, anti-oxidation, anti-coagulation, anti-tumor, and immunity-enhancing medicines or health products or food.
  • the beneficial effects of the present invention are reflected in: 1) through three times of salting out and alcohol precipitation, part of the protein (insoluble matter) in the enzymatic hydrolysis supernatant is effectively removed, so as to prevent the sample from clogging the resin pores in the chromatography column and reducing the loading capacity of the chromatography column, Thereby ensuring the repeatability of the elution behavior of the chromatography column, improving the service life of the filler, and at the same time improving the purity of the polysaccharide and reducing the residual protein in the sample; 2) Through anion exchange column chromatography, the crude sea cucumber polysaccharide can be effectively purified.
  • Sea cucumber fucoidan is a polyanionic polysaccharide, which is in a strongly charged polyanionic state in solution. In theory, it and anion exchange resin can be separated from other non-polyanionic substances by anion exchange resin.
  • the yield of sea cucumber polysaccharide can reach 3.6-4.3%, and the polysaccharide content can reach more than 65%.
  • Extraction of crude sea cucumber polysaccharide Weigh 3kg of dry rough sea cucumber, pulverize it into coarse particles, add 12 times the weight of sea cucumber in pure water, and use papain for enzymatic hydrolysis. 5. The temperature is 60° C. and the time is 3 hours; the subsequent operation is the same as that in Example 3, and 303.9 g of crude sea cucumber polysaccharide is obtained with a yield of 10.13%; 92.7 g of sea cucumber polysaccharide is obtained, and the yield of sea cucumber polysaccharide is 3.09%.
  • Extraction of crude sea cucumber polysaccharide take 3kg of dry rough sea cucumber, pulverize it into coarse particles, add 12 times the pure water of the sea cucumber weight, and use papain for enzymolysis.
  • the enzymolysis conditions are: the amount of enzyme added is 3% of the sea cucumber weight, pH6.5, temperature 60°C, time 6h; after enzymolysis, boil to inactivate the enzyme, centrifuge at 14000rpm in a tube centrifuge, take the supernatant, and concentrate under reduced pressure (temperature 50°C, vacuum degree ⁇ -0.085MPa) to the original 1/5 of the volume, centrifuge the tube centrifuge again at 14,000 rpm, and take the supernatant as the crude sea cucumber polysaccharide solution.
  • Example 1 Take the sea cucumber polysaccharide obtained in Example 1, Example 2, Example 3, Comparative Example 1 and Comparative Example 2 respectively, and measure its total polysaccharide content by the phenol-sulfuric acid method; use the Folin-phenol method to measure the residual protein content; its detection results See Table 1.
  • Table 1 The yield, total polysaccharide content and residual protein content of the sea cucumber polysaccharides prepared by the examples and comparative examples
  • Example 1 to Example 3 the polysaccharide yield and content obtained in Example 1 to Example 3 are all relatively high, and with the increase of enzyme addition, the polysaccharide yield and content also slightly increase;
  • the difference between the preparation methods of Example 3 and Comparative Example 1 is mainly the length of the enzymatic hydrolysis time, which can be obtained from the data in Table 1: extending the enzymatic hydrolysis time within a certain range can improve the yield and content of the polysaccharide; (2) Example 1 to implementation Example 4.
  • Comparative Example 1 has the operation steps of salting out and alcohol precipitation, and the residual protein content in the sea cucumber polysaccharide samples is all about 0.2%, while Comparative Example 2 lacks the step of salting out and alcohol precipitation, and its residual protein content is about 0.2%. It is higher (1.97%), so the addition of the salting-out alcohol precipitation step can effectively reduce the residual protein in the sample while ensuring the repeatability of the elution behavior of the chromatography column and improving the service life of the filler.

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Abstract

一种海参多糖的制备方法，步骤如下：(1)取干燥海参，粉碎成粗颗粒，加水酶解后，离心取上清液，上清液减压浓缩至原体积的1/5，再离心(离心条件同上)取上清液；上清液经三次盐析醇沉，沉淀再用无水乙醇洗涤两次，挥干乙醇后得海参多糖粗品；(2)将海参多糖粗品用纯水溶解，上阴离子层析柱(OH -)，用纯水及梯度浓度的NaCl溶液洗脱，收集NaCl洗脱液；(3)将上述NaCl洗脱液进行超滤脱盐，超滤截留液减压浓缩并冷冻干燥，即为海参多糖。海参多糖可用于制备抗高血脂症、抗氧化、抗凝血、抗肿瘤、提升免疫力等药物或保健品，具有预防血栓的功效，对老年人、血栓高风险人群具有良好的辅助预防作用。

Description

一种海参多糖的制备方法与应用 技术领域
本发明涉及一种海参多糖及其制备方法与应用。
技术背景
海参是传统滋补中药材，对海参的功效认可度高，但加工方法与服用方法简单而单一，吸收效果不能保障，容易造成营养成分的浪费。
海参是我国传统保健品,具有极高的保健及营养价值,也是海洋功能食品的重要研究对象。海参多糖作为海参的重要活性成分之一,具有抗高血脂症、抗氧化、抗凝血、抗肿瘤、提升免疫力等多种生物活性。海参多糖的研究主要集中在海参体壁,体壁多糖包括海参硫酸软骨素(SC-CHS)和海参岩藻聚糖硫酸酯(SC-FUC)2种组分(张健等，海参多糖制备、化学分析及生物活性研究进展[J]，食品安全质量检测学报，2018，9(08):1730-1736)。
海参岩藻聚糖硫酸酯(SC-FUC)是海参的主要功效成分之一，属于岩藻聚糖硫酸酯类化合物，具有多种生理调节功能，在功能食品的开发方面展现出良好的应用前景。目前关于SC-FUC结构研究的报道仍十分稀少，结构信息的匮乏限制了SC-FUC的深入研究及开发利用。
现有的海参多糖传统提取工艺可分为五类：酸法、碱法、酶法、盐法和热水浸提法等，其基本原理都是根据海产品的特性，改变蛋白的外界环境，从而把多糖分离出来。由于上述方法在提取过程中会使用大量的淡水和酸碱盐，属于高能耗产业，排放的酸碱盐对周围的环境产生一定的生态影响，不符合绿色可持续性经济发展模式，且提取工艺相对粗放。
发明内容
本发明所要解决的技术问题是提供一种结合生物酶解技术、盐析醇沉法、柱层析纯化脱色以及膜分离技术脱盐来提取海参多糖的方法，既能减少能源的消耗，又能提高海参多糖的提取率。
为实现上述目的，本发明提供以下技术方案：
一种制备海参多糖的方法，包括以下步骤：
1)海参多糖粗品的提取：将海参粉碎，加入纯水，用木瓜蛋白酶酶解，酶解完毕后，煮沸灭活酶，离心，取上清液减压浓缩至原体积的20％-25％，重复离心，取上清液经三次盐析 醇沉处理，得海参多糖粗品；
2)将上述海参多糖粗品用纯水溶解，上阴离子交换层析柱(OH -)，依次用纯水及梯度浓度的NaCl溶液洗脱，收集用NaCl溶液洗脱时的洗脱液；
3)将上述收集的洗脱液进行超滤脱盐处理，收集超滤截留液，减压浓缩并冷冻干燥，即得海参多糖。
其中步骤1)中，将干燥海参，粉碎(粉碎成粗颗粒)，加入海参重量8-12倍的纯水，用木瓜蛋白酶酶解。海参粉碎为粗颗粒，利于反应进行。
其中步骤1)中，酶解条件为：木瓜蛋白酶添加量为海参重量的1％-3％，pH6.0-7.0，温度50℃-60℃，时间4-6h。优选地，酶解条件为：木瓜蛋白酶添加量为海参重量的3％，pH 6.5，温度60℃，时间6h。
其中步骤1)中，离心速度为4000-15000rpm。更优选地，离心速度为15000rpm。
其中步骤1)中，盐析醇沉处理包括步骤：上清液中加入乙酸钾至终浓度为0.5M，加入乙醇至终浓度为60％(v/v)，离心，得沉淀A；沉淀A用纯水溶解，加入乙酸钾至终浓度为1.0M，加入乙醇至终浓度为40％(v/v)，离心，得沉淀B；沉淀B用纯水溶解，加入乙酸钾至终浓度为1.0M，加入乙醇至终浓度为40％(v/v)，离心，得沉淀C；沉淀C用无水乙醇洗涤，挥干乙醇，得海参多糖粗品。
其中步骤2)中，NaCl溶液的浓度为0.5-3.0mol/L。
其中步骤2)中，将海参多糖粗品用纯水溶解，上D204型阴离子层析柱(OH -)，依次分别用2-3倍柱体积的去离子水、2-3倍柱体积的0.3-0.6mol/L的NaCl溶液、2-3倍柱体积的0.9-1.2mol/L的NaCl溶液、2-3倍柱体积的1.8-2.4mol/L的NaCl溶液梯度洗脱，洗脱流速为2BV/h，收集用1.8-2.4mol/L NaCl溶液洗脱时的洗脱液。然后用约3mol/L NaCl溶液洗脱以清洗层析柱。优选的，依次分别用3倍柱床体积的纯水和0.5、1.0、2.0mol/L NaCl溶液梯度洗脱，收集用2.0mol/L NaCl溶液洗脱时的洗脱液。然后用约3mol/L NaCl溶液洗脱以清洗层析柱。
其中步骤3)中，将步骤2)收集的洗脱液经5KD中空纤维柱超滤脱盐处理，收集超滤截留液，于50℃-60℃，真空度≤-0.085MPa条件下减压浓缩，冷冻干燥，得海参多糖。
优选地，步骤3)中，超滤条件为：回流压≤0.15MPa，超滤截留液体积至超滤前二分之一时，开始补水，补水至超滤前的液体体积数；超滤行为持续至超滤截留液、透过液中Cl -均为阴性。
本发明的另一目的是提供一种上述任意方法制备的海参多糖。
本发明的再一个目的是上述任意方法制得的海参多糖在制备抗高血脂症、抗氧化、抗凝血、抗肿瘤、提升免疫力的药品或保健品或食品中的应用。
本发明的有益效果体现在：1)通过三次盐析醇沉，有效去除酶解上清液中的部分蛋白(不溶物)，避免样品堵塞层析柱中的树脂孔隙和降低层析柱载样量，从而保障层析柱洗脱行为的重复性、提升填料的使用寿命，同时能提高多糖纯度及降低样品中的残留蛋白；2)通过阴离子交换柱层析，有效纯化海参多糖粗品。海参岩藻聚糖是聚阴离子多糖，它在溶液中呈强电荷的聚阴离子状态，理论上，它与阴离子交换树脂可以用阴离子交换树脂实现与其他非聚阴离子物质分离。海参多糖得率可达到3.6～4.3％，多糖含量可达65％以上。
具体实施方式
通过实施例进一步详细描述本发明。以下实施例仅用于举例说明的目的，不应理解为对本发明保护范围的任意限制。除非特别说明，实施例中所用的材料、试剂和仪器均为普通市售或常规产品。
D204型阴离子层析柱：郑州勤实科技有限公司
实施例1海参多糖的制备
1)海参多糖粗品的提取：称取3kg干燥糙海参，粉碎成粗颗粒，加入海参重量8倍的纯水，用木瓜蛋白酶酶解，酶解条件为：酶添加量为海参重量的1％，pH6.0，温度50℃，时间6h；酶解完毕后，煮沸灭活酶，管式离心机15000rpm离心，取上清液，减压浓缩(温度50℃、真空度≤-0.085MPa)至原体积的20％，再次管式离心机15000rpm离心，取上清液；上清液第一次盐析醇沉：上清液中加入乙酸钾(使乙酸钾终浓度为0.5M)，以及乙醇(使乙醇终浓度为60％)，管式离心机离心取沉淀；沉淀用适量纯水溶解，进行第二次盐析醇沉：上清液中加入乙酸钾(使乙酸钾终浓度为1.0M)，以及乙醇(使乙醇终浓度为40％)，管式离心机离心取沉淀；沉淀用适量纯水溶解，进行第三次盐析醇沉：上清液中加入乙酸钾(使乙酸钾终浓度为1.0M)，以及乙醇(使乙醇终浓度为40％)，管式离心机离心取沉淀，沉淀再用无水乙醇洗涤两次，挥干乙醇后得362.4g海参多糖粗品，得率为12.08％。
2)将上述海参多糖粗品用纯水溶解，上D204型阴离子层析柱(OH -)，依次用3倍柱床体积的纯水和0.4、0.9、2.1、3.0mol/L NaCl溶液梯度洗脱，收集用2.1mol/L NaCl溶液洗脱时的洗脱液。
3)将上述收集的洗脱液进行5KD的中空纤维柱超滤脱盐处理，收集超滤截留液，减压浓缩(温度50℃、真空度≤-0.085MPa)，并冷冻干燥，得108.8g海参多糖，海参多糖得率 为3.63％。
实施例2海参多糖的制备
1)海参多糖粗品的提取：称取3kg干燥糙海参，粉碎成粗颗粒，加入海参重量10倍的纯水，用木瓜蛋白酶酶解，酶解条件为：酶添加量为海参重量的2％，pH7.0，温度55℃，时间5h；酶解完毕后，煮沸灭活酶，管式离心机12000rpm离心，取上清液，减压浓缩(温度50℃、真空度≤-0.085MPa)至原体积的25％，再次管式离心机16000rpm离心，时间10-15min，取上清液；上清液第一次盐析醇沉：上清液中加入乙酸钾(使乙酸钾终浓度为0.5M)，以及乙醇(使乙醇终浓度为60％)，管式离心机离心取沉淀；沉淀用适量纯水溶解，进行第二次盐析醇沉：上清液中加入乙酸钾(使乙酸钾终浓度为1.0M)，以及乙醇(使乙醇终浓度为40％)，管式离心机离心取沉淀；沉淀用适量纯水溶解，进行第三次盐析醇沉：上清液中加入乙酸钾(使乙酸钾终浓度为1.0M)，以及乙醇(使乙醇终浓度为40％)，管式离心机离心取沉淀，沉淀再用无水乙醇洗涤两次，挥干乙醇后得370.1g海参多糖粗品，得率为12.34％。
2)将上述海参多糖粗品用纯水溶解，上D204型阴离子层析柱(OH -)，依次用3倍柱床体积的纯水和0.6、1.1、1.9、3.0mol/L NaCl溶液梯度洗脱，收集用1.9mol/L NaCl溶液洗脱时的洗脱液。
3)将上述收集的洗脱液进行5KD的中空纤维柱超滤脱盐处理，收集超滤截留液，减压浓缩(温度50℃、真空度≤-0.085MPa)，冷冻干燥，得115.3g海参多糖，海参多糖得率为3.84％。
实施例3海参多糖的制备
1)海参多糖粗品的提取：称取3kg干燥糙海参，粉碎成粗颗粒，加入海参重量12倍的纯水，用木瓜蛋白酶酶解，酶解条件为：酶添加量为海参重量的3％，pH6.5，温度60℃，时间6h；酶解完毕后，煮沸灭活酶，管式离心机14000rpm离心，取上清液，减压浓缩(温度50℃、真空度≤-0.085MPa)至原体积的20％，再次管式离心机14000rpm离心，取上清液；上清液第一次盐析醇沉：上清液中加入乙酸钾(使乙酸钾终浓度为0.5M)，以及乙醇(使乙醇终浓度为60％)，管式离心机离心取沉淀；沉淀用适量纯水溶解，进行第二次盐析醇沉：上清液中加入乙酸钾(使乙酸钾终浓度为1.0M)，以及乙醇(使乙醇终浓度为40％)，管式离心机离心取沉淀；沉淀用适量纯水溶解，进行第三次盐析醇沉：上清液中加入乙酸钾(使乙酸钾终浓度为1.0M)，以及乙醇(使乙醇终浓度为40％)，管式离心机离心取沉淀，沉淀再用无水乙醇洗涤两次，挥干乙醇后得420.6g海参多糖粗品，得率为14.02％。
2)将上述海参多糖粗品用纯水溶解，上D204型阴离子层析柱(OH -)，依次用3倍柱 床体积的纯水和0.5、1.0、2.0、3.0mol/L NaCl溶液梯度洗脱，收集用2.0mol/L NaCl溶液洗脱时的洗脱液。
3)将上述收集的洗脱液进行5KD的中空纤维柱超滤脱盐处理，收集超滤截留液，减压浓缩(温度50℃、真空度≤-0.085MPa)并冷冻干燥，得130.1g海参多糖，海参多糖得率为4.34％。
对比例1海参多糖的制备
海参多糖粗品的提取：称取3kg干燥糙海参，粉碎成粗颗粒，加入海参重量12倍的纯水，用木瓜蛋白酶酶解，酶解条件为：酶添加量为海参重量的3％，pH6.5，温度60℃，时间3h；后续操作同实施例3，得303.9g海参多糖粗品，得率为10.13％；得92.7g海参多糖，海参多糖得率为3.09％。
对比例2海参多糖的制备
1)海参多糖粗品的提取：称取3kg干燥糙海参，粉碎成粗颗粒，加入海参重量12倍的纯水，用木瓜蛋白酶酶解，酶解条件为：酶添加量为海参重量的3％，pH6.5，温度60℃，时间6h；酶解完毕后，煮沸灭活酶，管式离心机14000rpm离心，取上清液，减压浓缩(温度50℃、真空度≤-0.085MPa)至原体积的1/5，再次管式离心机14000rpm离心，取上清液作为海参多糖粗品溶液。
2)将上述海参多糖粗品溶液上D204型阴离子层析柱(OH -)，依次用3倍柱床体积的纯水和0.5、1.0、2.0、3.0mol/L NaCl溶液梯度洗脱，收集用2.0mol/L NaCl溶液洗脱时的洗脱液；
3)将上述收集的洗脱液进行5KD的中空纤维柱超滤脱盐处理，收集超滤截留液，减压浓缩(温度50℃、真空度≤-0.085MPa)并冷冻干燥，得117.3g海参多糖，海参多糖得率为3.91％。
分别取实施例1、实施例2、实施例3、对比例1、对比例2所得的海参多糖，采用苯酚-硫酸法测其总多糖含量；采用Folin-酚法测定残留蛋白含量；其检测结果见表1。
表1：实施例及对比例制备所得海参多糖的得率、总多糖含量及残留蛋白含量
Figure PCTCN2020119685-appb-000001
Figure PCTCN2020119685-appb-000002
从表1中可以看出，(1)实施例1至实施例3所得的多糖得率及含量均较高，随着酶添加量的增加，多糖得率及含量也随之略有提高；实施例3与对比例1的制备方法区别主要在酶解时间的长短，由表1数据可得：在一定范围内延长酶解时间能够提高多糖的得率和含量；(2)实施例1至实施例4、对比例1的制备方法均有盐析醇沉的操作步骤，其海参多糖样品中残留蛋白含量均在0.2％左右，而对比例2缺少了盐析醇沉的步骤，其残留蛋白含量较高(1.97％)，因此盐析醇沉步骤的加入在保障层析柱洗脱行为重复性、提升填料使用寿命的同时，能有效降低样品中的残留蛋白。

Claims (10)

  1. 一种制备海参多糖的方法，包括以下步骤：
    1)海参多糖粗品的提取：将海参粉碎，加入纯水，用木瓜蛋白酶酶解，酶解完毕后，煮沸灭活酶，离心，取上清液减压浓缩至原体积的20％-25％，重复离心，取上清液经三次盐析醇沉处理，得海参多糖粗品；
    2)将上述海参多糖粗品用纯水溶解，上阴离子交换层析柱(OH -)，依次用纯水及梯度浓度的NaCl溶液洗脱，收集用NaCl溶液洗脱时的洗脱液；
    3)将上述收集的洗脱液进行超滤脱盐处理，收集超滤截留液，减压浓缩并冷冻干燥，即得海参多糖。
  2. 如权利要求1所述的方法，其中步骤1)中，将干燥海参粉碎，加入海参重量8-12倍的纯水，用木瓜蛋白酶酶解；酶解条件为：木瓜蛋白酶添加量为海参重量的1％-3％，pH6.0-7.0，温度50℃-60℃，时间4-6h；离心速度为4000-15000rpm。
  3. 如权利要求2所述的方法，其中步骤1)中，酶解条件为：木瓜蛋白酶添加量为海参重量的3％，pH 6.5，温度60℃，时间6h。
  4. 如权利要求1所述的方法，其中步骤1)中，盐析醇沉处理包括步骤：上清液中加入乙酸钾至终浓度为0.5M，加入乙醇至终浓度为60％(v/v)，离心，得沉淀A；沉淀A用纯水溶解，加入乙酸钾至终浓度为1.0M，加入乙醇至终浓度为40％(v/v)，离心，得沉淀B；沉淀B用纯水溶解，加入乙酸钾至终浓度为1.0M，加入乙醇至终浓度为40％(v/v)，离心，得沉淀C；沉淀C用无水乙醇洗涤，挥干乙醇，得海参多糖粗品。
  5. 如权利要求1所述的方法，其中步骤2)中，NaCl溶液的浓度为0.5-3.0mol/L。
  6. 如权利要求1所述的方法，其中步骤2)中，将海参多糖粗品用纯水溶解，上D204型阴离子层析柱，依次分别用2-3倍柱体积的去离子水、2-3倍柱体积的0.3-0.6mol/L的NaCl溶液、2-3倍柱体积的0.9-1.2mol/L的NaCl溶液、2-3倍柱体积的1.8-2.4mol/L的NaCl溶液梯度洗脱，洗脱流速为2BV/h，收集用1.8-2.4mol/L NaCl溶液洗脱时的洗脱液。
  7. 如权利要求1所述的方法，其中步骤3)中，将步骤2)收集的洗脱液经5KD中空纤维柱超滤脱盐处理，收集超滤截留液，于50℃-60℃，真空度≤-0.085MPa条件下减压浓缩，冷冻干燥，得海参多糖。
  8. 如权利要求1所述的方法，其中步骤3)中，超滤条件为：回流压≤0.15MPa，超滤截留液体积至超滤前二分之一时，开始补水，补水至超滤前的液体体积数；超滤行为持续至超滤截留液、透过液中Cl -均为阴性。
  9. 一种根据权利要求1-8任一项所述的方法制备的海参多糖。
  10. 如权利要求9所述的海参多糖在制备抗高血脂症、抗氧化、抗凝血、抗肿瘤、提升免疫力的药品或保健品或食品中的应用。
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