WO2012013112A1 - 盐析萃取海参中有效成分的方法 - Google Patents

盐析萃取海参中有效成分的方法 Download PDF

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WO2012013112A1
WO2012013112A1 PCT/CN2011/076780 CN2011076780W WO2012013112A1 WO 2012013112 A1 WO2012013112 A1 WO 2012013112A1 CN 2011076780 W CN2011076780 W CN 2011076780W WO 2012013112 A1 WO2012013112 A1 WO 2012013112A1
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sea cucumber
phase
extraction
salt
low molecular
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French (fr)
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修志龙
杨向春
惠欢庆
李晓晖
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Dalian University of Technology
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Dalian University of Technology
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/50—Molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives

Definitions

  • the invention belongs to the technical field of deep processing of seafood, and relates to a method for extracting active ingredients from sea cucumber by salting out, in particular to a method for extracting active ingredients of sea cucumber by two-phase and three-liquid phase extraction.
  • Sea cucumber is a kind of seafood treasure that people are familiar with. It is called 'sea ginseng'. It has high nutritional value and medicinal value.
  • the sea cucumber is rich in amino acids, essential fatty acids, vitamins and chemical elements.
  • a variety of nutrients also contains a variety of biologically active substances, such as sea cucumber saponins, sea cucumber polysaccharides, sea cucumber collagen, polyunsaturated fatty acids, cerebrosides and sea cucumber peptides produced by sea cucumber enzymatic hydrolysis.
  • These ingredients have high nutritional value and medicinal value, such as: essential fatty acids play an important role in enhancing human immunity and human brain function; sea cucumber saponins have anti-tumor, anti-fungal, anti-radiation, anti-protozoal effects and immunity Regulation and anti-fatigue effects; sea cucumber polysaccharide has anti-coagulation, anti-tumor, immune regulation, anti-aging and other biological activities; sea cucumber protein has blood, nourishing , It protects the gastric mucosa and anti-ulcer effect, as well as biological functions such as anti-allergic, blood pressure lowering, cholesterol lowering, anti-aging, promoting wound healing and promoting calcium absorption. Because various ingredients have high nutritional value and medicinal value, they are widely used in medicine, health care, food and other fields.
  • the extraction of the active ingredients of sea cucumber is mostly carried out by using fresh sea cucumber as raw material, extracting sea cucumber polysaccharide by enzymatic hydrolysis and alkali extraction and alcohol precipitation, or combining acid precipitation with multi-stage alcohol precipitation and extracting protein by enzymatic hydrolysis combined with salting out.
  • These methods are all for the extraction and separation of a certain sea cucumber active substance (such as protein or polysaccharide).
  • the effective active substances of sea cucumber are not comprehensively considered, which is easy to cause waste of precious resources, and the complicated extraction and separation method leads to high product cost.
  • the sea cooking cooking liquid contains almost the same composition as the sea cucumber itself, such as The extraction of these active ingredients with 10-30 g/l protein and 150-300 g/l crude polysaccharide can undoubtedly significantly increase the added value of sea cucumber products.
  • Zhao Qiancheng et al. obtained the crude protein and polysaccharide from the sea cooking steam by the method of isoelectric point settlement and graded alcohol precipitation
  • Yuan Wenpeng et al. used alcohol precipitation technology to recover polysaccharide from sea cucumber cooking juice. (CN101343332)
  • the above method is only for one or two active substances, and other active ingredients of sea cucumber are easy to be lost in the process of extraction and separation;
  • the alcohol precipitation technology has a large amount of ethanol, a long production cycle, and a large loss of active components, which increases the difficulty of purification and purification in the later stage.
  • the multi-phase salting-out extraction technology is an extraction and separation method capable of simultaneously extracting various active ingredients, simple operation, mild conditions, continuous operation, and easy amplification.
  • the novel aqueous two-phase extraction technology formed by hydrophilic low molecular organic substances and salts has the advantages of low price, easy solvent recovery and short phase separation time, so bio-based chemicals, amino acids, antibiotics, natural products, traditional Chinese medicine ingredients, etc. The extraction has been widely used.
  • the research team where the applicant is located has done a lot of research and development work (Chinese patent: CN 101531652A ; CN101012151A ; Biotechnology Letters , 2008 , 30 : 2079-2084 ; 2009, 31: 371-376).
  • the three-liquid phase system formed by the hydrophobic organic substance/hydrophilic low molecular organic substance/salt can simultaneously extract two or more active ingredients having a large difference in polarity. Simple operation, mild conditions, short phase separation time, etc.
  • the applicant's research group applied the technology to the extraction and separation of natural products and achieved the desired effect (Chinese patent: CN101637667; Process Biochemistry, 2010, 45: 752-756 ) .
  • the present invention attempts to extract the active ingredients in sea cucumber by using a multi-phase salting-out extraction system.
  • the invention provides a novel aqueous two-phase system formed by a hydrophilic low molecular organic substance/salt and a hydrophobic organic substance/hydrophilic low molecular organic substance/salt/the problem of long production cycle and large solvent consumption.
  • the water three-liquid phase system simultaneously extracts and separates various active ingredients from the sea cooking cooking liquor.
  • a method for salting out extraction and separating active ingredients of sea cucumber the operation steps are as follows: In the sea cucumber extract, some electrolytes and common organic solvents are directly added, and the hydration of the salt ions is used to form an organic solvent. Salt multiphase salting out extraction system. According to the difference in solubility, the distribution of the active ingredients with different polarities in the sea cucumber is different in each phase, thereby achieving separation and concentration of the active ingredients.
  • salt and hydrophilic low molecular organics Adding to sea cucumber extract in a certain proportion, stirring evenly, standing or centrifuging to form a double aqueous phase: sea cucumber alcohol soluble active ingredient is concentrated in the upper phase, protein and polysaccharide are mainly distributed to the solid phase between the upper phase and the lower phase Medium, heavy metals are enriched in the lower phase;
  • the hydrophilic low molecular organic substance and the hydrophobic organic substance are added to the sea cucumber extract liquid in a certain proportion, stirred uniformly, and left standing or centrifuged to form a three-liquid salting out extraction system: Sea cucumber oil is enriched in the upper phase, and a small amount of soluble protein and polysaccharide are distributed in the middle phase. Most of the protein and polysaccharide are distributed in the solid phase between the middle phase and the lower phase, while heavy metals are enriched in the lower phase.
  • the mass percentage of the salt in the aqueous two-phase system is 10 ⁇ 30%, and the mass percentage of the hydrophilic low molecular organic substance is 15 ⁇ 40%.
  • the extraction temperature is 15 ⁇ 40 °C
  • the time of stirring and standing phase separation is 0.1 ⁇ 4.0 h, or it can be centrifuged at 2000 ⁇ 8500 rpm for 5 ⁇ 20 min.
  • the mass percentage of the salt in the three liquid phase system is 10 ⁇ 30%
  • the mass percentage of the hydrophilic low molecular organic matter is 15 ⁇ 40%
  • the mass percentage of the hydrophobic organic matter is 10 ⁇ 40%.
  • the applicable sea cucumber extract refers to a liquid containing the active ingredient of sea cucumber, including fresh sea cucumber juice or a concentrated liquid thereof, or a sea cooking cooking liquid or a concentrated liquid thereof.
  • the sea cooking cooking liquid refers to the waste liquid obtained by cooking sea cucumber.
  • the hydrophilic low molecular organic substances selected are ethanol, ethylene glycol, n-propanol, isopropanol, 1,2-propanediol, 1 , 3- One of propylene glycol, n-butanol, isobutanol, tert-butanol, 1,2-butanediol, 1, 3-butanediol, 1, 4-butanediol, 2, 3-butanediol, acetonekind or several combinations;
  • the selected salts are sodium chloride, lithium chloride, ammonium sulfate, sodium sulfate, sodium carbonate, potassium carbonate, sodium acetate, potassium acetate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium citrate, citric acid.
  • the hydrophobic organic material selected is methyl acetate, ethyl a
  • the extraction operation may be one extraction, multiple extraction, continuous extraction or countercurrent extraction.
  • the invention has the beneficial effects that compared with the prior art extraction and separation method, the present invention utilizes a multi-phase salting-out extraction system to simultaneously extract and separate a plurality of active components with different polarities, and obtains sea cucumber oil and alcohol-soluble active ingredients ( With ethanol as extractant, a variety of products such as sea cucumber wine, crude protein and polysaccharide can be prepared to complete the preliminary purification of the target product.
  • the content of harmful heavy metal ions (arsenic, mercury, lead, cadmium) in the solid phase lyophilized powder obtained after extraction is significantly reduced. Most of the beneficial ions (calcium, iron, magnesium, zinc) remain.
  • the whole production process is simple in operation, low in production cost, short in cycle, mild in extraction conditions, cheap and easy to recover organic solvent, low in mass transfer, fast in mass separation, and easy to industrialize.
  • the product meets the requirements of food safety.
  • the sea-based work cooking liquid concentrate used in the examples was obtained by the following method.
  • the filter is used for coarse filtration of sea cucumber cooking waste liquid, the filtrate is centrifuged, and the supernatant is subjected to ultrafiltration concentration, desalting, sterilization, and enzyme elimination, and then centrifuged, and finally concentrated by three effects to obtain sea cucumber cooking and concentration. liquid.
  • the concentration of the protein in the examples was determined by Kjeldahl method (GB 5009.5-85); the polysaccharide was determined by the DNS method; GB/T 5009.6 - 2003 ); metal ions are detected by atomic absorption spectrometry according to national standard: F e , Mg (GB/T 5009.90-2003) , Zn (GB/T 5009.14-2003), Cd (GB/T 5009.15-2003), Se (GB/T 5009.93-2003), Pb (GB/T 5009.12-2003) , As (GB/T 23372-2009) , Hg (GB/T 5009.17-1996) , Ca (GB/T 5009.92-2003), Cu (GB/T 5009.13-2003).
  • Example 1 Extraction of Active Components from Sea Cucumber by Sodium Carbonate / Ethanol System
  • Step 1 Add anhydrous sodium carbonate and ethanol to the sea cooking concentrate to make the concentration reach 16% and 21% (w/w). Shake well, stir and place in a water bath at 37 °C for 10 min to form a double aqueous phase, and centrifuge at 5000 rpm for 10 min to complete the phase separation. Determination of the protein and polysaccharide of each phase indicates that there is Ninety percent of the sugar and 96.1% of the protein were partitioned in the solid phase.
  • Step 2 Take out the solid phase between the upper and lower phase interface layers and freeze-dry them to make a powder.
  • Step 3 The obtained powder sample is subjected to digestion treatment to measure metal ions, and the measurement results show that metal mercury, lead and arsenic are not detected; the content of cadmium is 2.865 mg/L, reduced to 60% of the original concentrated liquid dry powder; selenium content is 0.150 mg / L; calcium content is 2785 mg / L; copper content is 3.7 Mg/L; iron content is 95.6 mg/L; magnesium content is 867.5 mg/L; zinc content is 9.2 mg/L.
  • Step 1 Add anhydrous sodium carbonate and ethanol to the sea cooking concentrate to make the concentration reach 16% and 21% (w/w).
  • Step 2 The upper phase was taken out and the n-hexane was evaporated to obtain a fat.
  • the extraction rate of sea cucumber oil obtained by three-liquid extraction was 63.3%.
  • Step 1 Add ammonium sulfate (30% w/v) and tert-butanol (1:11.5) to the sea cooking concentrate, and participate in the cooking liquor.
  • Step 2 Take 187 ml of the upper phase of the aqueous two phase and remove the t-butanol by rotary evaporation at 37 ° C. Sea cucumber alcohol soluble active ingredient.
  • Step 3 Take out the solid phase between the upper and lower phase interface layers and freeze-dry them to make a powder.
  • Step 4 The prepared powder sample is subjected to perchloric acid and concentrated nitric acid. Digestion treatment, determination of metal ions, the results show that the content of harmful heavy metal cadmium is 0.620 mg / L, reduced to 13 % of the original concentrated liquid dry powder.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Polymers & Plastics (AREA)
  • Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Zoology (AREA)
  • Mycology (AREA)
  • Extraction Or Liquid Replacement (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Peptides Or Proteins (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

本发明公开了一种盐析萃取海参中有效成分的方法，属于海产品深加工技术领域。向海参提取液中，直接加入某些电解质和普通有机溶剂，利用盐离子的水化作用，形成有机溶剂/盐多相盐析萃取体系。根据溶解度差异，海参中极性不同的有效成分在各相中的分配不同，实现有效成分的分离与浓縮。将盐与亲水性低分子有机物按一定比例加入到海参提取液中，搅拌均匀，静置或离心，形成双水相；另外将盐、亲水性低分子有机物以及疏水性有机物按一定比例加入到海参提取液中，搅拌均匀，静置或离心，形成三液相盐析萃取体系。本发明开发了一种同时提取与分离海参多种有效成分、操作简便、条件温和、成本低廉、并有效降低重金属毒害作用的新型盐析萃取方法。

Description

盐析萃取海参中有效成分的方法
技术领域
本发明属于海产品深加工技术领域，涉及一种从海参中盐析萃取有效成分的方法，特别涉及双水相和三液相萃取海参有效成分的方法。
背景技术
海参是人们熟悉的一种海产珍品，被称为 ' 海中人参 ' ，具有很高的营养价值和药用价值。海参体内不但富含氨基酸、必需脂肪酸、维生素和化学元素等人体所需的 50 多种营养成分，还含有多种生物活性物质，如海参皂苷、海参多糖、海参胶原蛋白、多不饱和脂肪酸、脑苷脂类以及海参经酶解产生的海参肽等。这些成分均有很高的营养价值和药用价值，如：必需脂肪酸对增强人体免疫力和人脑的机能具有重要作用；海参皂苷具有抗肿瘤、抗真菌、抗放射、抗原虫的作用以及免疫调节和抗疲劳等作用；海参多糖具有抗凝血、抗肿瘤、免疫调节、延缓衰老等多种生物活性；海参蛋白具有生血、养血 、 保护胃粘膜及抗溃疡作用，还有抗过敏、降血压、降胆固醇、抗衰老、促进伤口愈合和促进钙质吸收等生物学功能。由于各种成分均具有很高的营养价值和药用价值，因此在医药、保健、食品等领域有着广泛的应用。
目前海参有效成分的提取多采用鲜海参做原料，经酶解法和碱提醇沉法提取海参多糖，或用酸沉结合多级醇沉的方法以及利用酶解结合盐析的方法提取蛋白质。这些方法都是针对某种海参活性物质（如蛋白质或者多糖）进行提取分离，通常没有综合考虑海参其它的有效活性物质，容易造成珍贵资源浪费，加之提取分离方法复杂导致产品成本偏高。而海参加工蒸煮液中含有与海参自身几乎相同的组成，如 10-30 g/l 蛋白和 150-300 g/l 粗多糖，将这些有效成分提取出来无疑可以显著提高海参制品的附加值。
利用海参加工蒸煮液提取与分离有效成分已有一些报道。如 赵前程等用等电点沉降和分级醇沉的方法从海参加工蒸煮液中获得粗蛋白和多糖 （中国专利： CN101585873A ）；袁文鹏等人采用醇沉技术从海参煮汁中回收多糖 （ CN101343332 ）；姜玉宝研究了醇沉、脱盐、蒸馏等方法回收海参煮汁中的油 （ CN101473979A ）。 虽然 这些技术 能在一定程度上达到分离的目的，但也存在明显的缺陷 : 一是上述方法只针对一、二种活性物质，其它海参有效成分容易在提取分离过程中损失；二是用的较多的 醇沉技术乙醇用量大，生产周期长，活性成分损失多， 给后期纯化精制也增加了难度。
针对上述存在的问题，开发简易高效的 提取分离 海参多种有效成分的方法具有重要的实用价值。多相盐析萃取技术是一种能同时萃取多种有效成分、操作简单、条件温和、可连续操作、易于放大的提取分离方法。其中由亲水性低分子有机物与盐形成的新型双水相萃取技术具有价格低廉、溶剂回收容易、分相时间短等优势，因此在生物基化学品、氨基酸、抗生素、天然产物、中药成分等的提取中得到了广泛的应用。申请人所在的课题组做了大量的研发工作（中国专利： CN 101531652A ； CN 101012151A ； Biotechnology Letters ， 2008 ， 30 ： 2079-2084 ； 2009 ， 31 ： 371-376 ）。而 由疏水性有机物 / 亲水性低分子有机物 / 盐形成的三液相体系可同时萃取两种以上极性相差较大的有效成分， 操作简单、条件温和、分相时间短等。 申请人所在课题组将该技术应用于天然产物的提取分离，取得了理想的效果 （ 中 国专利： CN101637667 ； Process Biochemistry, 2010 ， 45: 752-756 ） 。但在海参有效成分的提取分离中尚无文献报道，本发明尝试应用多相盐析萃取体系萃取海参中的有效成分。
发明内容
为解决现有海参有效成分提取中存在的目标产物单一、其它有效成分损失大、成本高、操作过程 繁琐 、生产周期长、溶剂耗量大等问题，本发明提供了一种亲水性低分子有机物 / 盐形成的新型双水相体系及疏水性有机物 / 亲水性低分子有机物 / 盐 / 水三液相体系从海参加工蒸煮液中同时提取分离多种有效成分的方法。
本发明的技术方案如下：
一种盐析萃取分离海参有效成分的方法，其操作步骤如下： 向海参提取液中，直接加入某些电解质和普通有机溶剂，利用盐离子的水化作用，形成有机溶剂 / 盐多相盐析萃取体系。根据溶解度差异，海参中极性不同的有效成分在各相中的分配不同，从而实现有效成分的分离与浓缩。如：将盐与 亲水性低分子有机物 按一定比例加入到海参提取液中，搅拌均匀，静置或离心，形成双水相：海参醇溶性有效成分富集在上相，蛋白质和多糖主要分配到上相和下相之间的固相中，重金属则被富集在下相；另外将盐、 亲水性低分子有机物以及疏水性有机物 按一定比例加入到海参提取液中，搅拌均匀，静置或离心，形成三液相盐析萃取体系 ： 海参油脂富集在上相，少量可溶性蛋白和多糖分配在中相， 绝大部分 蛋白和多糖分配在中相与下相之间的固相中，同时重金属被富集在下相。
其中双水相体系中盐的质量百分数为 10~30% ， 亲水性低分子有机物的质量百分数为 15~40% ， 萃取温度为 15~40℃，搅拌、静置分相的时间为0.1~4.0 h ， 或者在 2000 ~ 8500 rpm 下离心 5 ~ 20 min ；三液相体系盐的质量百分数为 10~30% ， 亲水性低分子有机物的质量百分数为 15~40% ，疏水性有机物的质量百分数为 10~40% ，搅拌均匀、静置分相 0.1~4.0 h 或者在 2000 ~ 8500 rpm 下离心 5 ~ 20 min ， 萃取温度为 15~40 ℃。
所适用的海参提取液是指含有海参有效成分的液体，包括鲜海参汁液或其浓缩液，也可以是海参加工蒸煮液或其浓缩液。海参加工蒸煮液是指蒸煮海参所得废液。
所选用的 亲水性低分子有机物 为 乙醇、乙二醇、正丙醇、异丙醇、 1 ， 2- 丙二醇、 1 ， 3- 丙二醇、正丁醇、异丁醇、叔丁醇 、 1, 2- 丁二醇、 1, 3- 丁二醇、 1, 4- 丁二醇、 2, 3- 丁二醇、 丙酮中的一种或几种组合； 所选用的盐为氯化钠、氯化锂、硫酸铵、硫酸钠、碳酸钠、碳酸钾、醋酸钠、醋酸钾、磷酸钾、磷酸二氢钾、磷酸氢二钾、柠檬酸钠、柠檬酸钾、草酸钠、草酸钾中的一种或几种组合； 所选用的疏水性有机物为乙酸甲酯、乙酸乙酯、正己烷。
其中 萃取操作可以是一次萃取、多次萃取、连续萃取或逆流萃取。
本发明的有益效果是和现有的提取分离方法相比较，本发明利用多相盐析萃取体系对极性不同的多种有效成分进行同步提取与分离，获得了海参油、醇溶性有效成分（以乙醇为萃取剂可制作海参酒）、粗蛋白和多糖等多种产品，完成目标产物的初步纯化。经过萃取后获得的固相冷干粉中有害重金属离子（砷、汞、铅、镉）的含量显著降低， 有益离子（钙、铁，镁、锌）大部分保留。 整个生产工艺操作简单、生产成本低、 周期短、萃取条件温和、 有机溶剂廉价易回收、粘度低传质分相快、易于产业化 、产品符合食品安全的要求 。
具体实施方式
实施例中所用的海参加工蒸煮液浓缩液是通过下述方法得到。
用 100 目滤网对海参蒸煮废液进行粗过滤，将滤液离心，再将离心上清液进行超滤浓缩脱盐、灭菌、灭酶，之后再离心过滤，最后经三效浓缩后即得海参蒸煮浓缩液。
实施例中蛋白的浓度用凯氏定氮法 (GB 5009.5-85) 确定；多糖用 DNS 法测定；油脂按国标法（ GB/T 5009.6 - 2003 ）测定；金属离子按国标用原子吸收光谱法检测： F e 、 Mg (GB/T 5009.90-2003) ， Zn (GB/T 5009.14-2003) ， Cd (GB/T 5009.15-2003) ， Se (GB/T 5009.93-2003) ， Pb (GB/T 5009.12-2003) ， As (GB/T 23372-2009) ， Hg (GB/T 5009.17-1996) ， Ca (GB/T 5009.92-2003) ， Cu (GB/T 5009.13-2003) 。
实施例 1 ：碳酸钠 / 乙醇体系萃取海参中的有效成分
步骤 1 向海参加工蒸煮液浓缩液中加入无水碳酸钠和乙醇，使其浓度达到 1 6 ﹪和 21 ﹪（w/w） ，搅拌摇匀，放在 3 7 ℃ 水浴中 静止 10min 形成双水相，并在 5000rmp 下离心 10min ，使分相完全。测定各相的蛋白和多糖表明，有 90.3 ﹪的糖和96.1﹪的蛋白分配在固相。
步骤 2 取出上、下相界面层间的固相，将其冷冻干燥制成粉末。
步骤 3 将制得的粉末样品经消化处理，进行金属离子的测定，测定结果显示，金属汞、铅、砷未检出；镉的含量为 2.865 mg/L ，降低到原浓缩液冷干粉的 60 ﹪ ；硒的含量为 0.150 mg/L ； 钙的含量为 2785 mg/L ； 铜的含量为 3.7 mg/L ； 铁的含量为 95.6 mg/L ； 镁的含量为 867.5 mg/L ； 锌的含量为 9.2 mg/L 。
实施例 2 ：三液相萃取海参有效成分
步骤 1 向海参加工蒸煮液浓缩液中加入无水碳酸钠和乙醇，使其浓度达到 1 6 ﹪和 21 ﹪（w/w） ，再加入正己烷使其浓度达到无水碳酸钠、乙醇、正己烷总质量的 28 ﹪（w/w） ，搅拌摇匀，放在 3 7 ℃ 水浴中 静止 10min 形成三液相，并在 5000rmp 下离心 10min ，使其分相完全。测定各相的蛋白和多糖表明，有 96.9 ﹪的蛋白和71.2﹪的糖分配在固相。
步骤 2 取出上相将正己烷蒸发得到油脂，经三液相萃取得到的海参油提取率为 63.3 ﹪。
实施例 3 ：硫酸铵 / 叔丁醇体系萃取海参中有效成分
步骤 1 向海参加工蒸煮液浓缩液中加入硫酸铵（ 30 ﹪w/v ）和叔丁醇（ 1:1.5 ，海参加工蒸煮液 v ：叔丁醇 v ），搅拌摇匀，放在室温静止 0.5h 形成双水相。
步骤 2 将双水相的 187ml 上相取出，在 3 7 ℃ 下旋转蒸发除去叔丁醇，得到 海参醇溶性有效成分。
步骤 3 取出上、下相界面层间的固相，将其冷冻干燥制成粉末 。
步骤 4 将制得的粉末样品经高氯酸 、浓硝酸 消化处理，进行金属离子的测定，测定结果显示有害重金属镉的含量为 0.620 mg/L ，降低到原浓缩液冷干粉的 13 ﹪ 。

Claims (1)

1. 一种盐析萃取分离海参中有效成分的方法，其特征在于：
(1) 将盐与 亲水性低分子有机物 水溶液按比例直接加入到海参提取液中，搅拌均匀，静置分相或离心分相，形成双水相 : 海参醇溶性有效成分富集在上相，蛋白质和多糖主要分配到上相和下相之间的固相中，重金属则被富集在下相；双水相体系中盐的质量百分数为 10~30% ， 亲水性低分子有机物的质量百分数为 15~40% ， 萃取温度为 15~40℃，静置分相的时间为0.1~4.0h 或者在 2000 ~ 8500 rpm 下离心 5 ~ 20 min ；
(2) 将盐、 亲水性低分子有机物以及疏水性有机物 按比例加入到海参提取液中，搅拌均匀，静置或离心，形成三液相盐析萃取体系 ： 海参油脂富集在上相，少量可溶性蛋白和多糖分配在中相， 绝大部分 蛋白和多糖分配在中相与下相之间的固相中，同时重金属被富集在下相；
三液相体系中盐的质量分数为 10~30% ， 亲水性低分子有机物的质量百分数为 15~40% ，疏水性有机物的质量百分数为 10~40% ，静置分相 0.1~4.0 h 或者在 2000 ~ 8500 rpm 下离心 5 ~ 20 min ， 萃取温度为 15~40℃。
2. 根据权利要求 1 所述的一种盐析萃取分离海参有效成分的方法，其特征在于： 所述的海参提取液是指含有海参有效成分的液体，包括鲜海参汁液、鲜海参汁液浓缩液、海参加工蒸煮液或海参加工蒸煮液浓缩液。
3. 根据权利要求 1 或 2 所述的一种盐析萃取分离海参有效成分的方法，其特征在于：所选用的 亲水性低分子有机物 为 乙醇、乙二醇、正丙醇、异丙醇、 1 ， 2- 丙二醇、 1 ， 3- 丙二醇、正丁醇、异丁醇、叔丁醇 、 1, 2- 丁二醇、 1, 3- 丁二醇、 1, 4- 丁二醇、 2, 3- 丁二醇、 丙酮中的一种或几种组合。
4. 根据权利要求 1 或 2 所述的一种盐析萃取分离海参有效成分的方法，其特征在于：所选用的盐为氯化钠、氯化锂、硫酸铵、硫酸钠、碳酸钠、碳酸钾、醋酸钠、醋酸钾、磷酸钾、磷酸二氢钾、磷酸氢二钾、柠檬酸钠、柠檬酸钾、草酸钠、草酸钾中的一种或几种组合。
5. 根据权利要求 1 或 2 所述的 一种盐析萃取分离海参有效成分 的方法，其特征在于：所选用的疏水性有机物为乙酸甲酯、乙酸乙酯或正己烷。
6. 根据权利要求 1 或 2 所述的 一种盐析萃取分离海参 有效成分的方法，其特征在于：萃取操作是一次萃取、多次萃取、连续萃取或逆流萃取。
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