WO2023011102A1 - 亚麻籽粕中多种成分的亚临界复合溶剂萃取分离方法 - Google Patents

亚麻籽粕中多种成分的亚临界复合溶剂萃取分离方法 Download PDF

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WO2023011102A1
WO2023011102A1 PCT/CN2022/104399 CN2022104399W WO2023011102A1 WO 2023011102 A1 WO2023011102 A1 WO 2023011102A1 CN 2022104399 W CN2022104399 W CN 2022104399W WO 2023011102 A1 WO2023011102 A1 WO 2023011102A1
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extraction
separation method
subcritical
flaxseed
linseed
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French (fr)
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李颖
黄静
汪勇
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暨南大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0288—Applications, solvents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/028—Flow sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0292—Treatment of the solvent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
    • B01D15/1871—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B01D15/426—Specific type of solvent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D3/00—Distillation or related exchange processes in which liquids are contacted with gaseous media, e.g. stripping
    • B01D3/08—Distillation or related exchange processes in which liquids are contacted with gaseous media, e.g. stripping in rotating vessels; Atomisation on rotating discs
    • B01D3/085—Distillation or related exchange processes in which liquids are contacted with gaseous media, e.g. stripping in rotating vessels; Atomisation on rotating discs using a rotary evaporator
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • 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
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/006—Refining fats or fatty oils by extraction
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the invention belongs to the field of extraction and separation of plant effective components, and in particular relates to a subcritical composite solvent extraction and separation method for various active components in linseed meal.
  • Flaxseed oil is rich in ⁇ -linolenic acid, VE and other nutrients.
  • the flaxseed cake obtained after pressing still contains residual oil, as well as active ingredients such as lignans, polysaccharides (gums) and proteins.
  • active ingredients such as lignans, polysaccharides (gums) and proteins.
  • linseed meal is usually only used as animal feed, fertilizer or waste disposal, and the resource is underutilized.
  • flaxseed products in my country are mainly flaxseed oil, which is single and homogeneous, and the development of flaxseed functional food ingredients has gradually become a hot spot.
  • Oils and fats are produced by pressing, solvent extraction and supercritical CO2 extraction.
  • the pressing method has a low yield and limited processing capacity.
  • Solvent extraction is often used to make up for the defects, but the quality of the oil obtained by this method is different, and there are Risk of solvent residues.
  • the emerging supercritical CO 2 extraction method has a high yield and good quality, but the industrial production cost is relatively high, and professional personnel are required to operate it.
  • flax lignans exist in flaxseed hulls, and the extraction processes are mainly divided into organic solvent extraction, microwave-assisted extraction and ultrasonic-assisted extraction.
  • the organic solvent method is currently the main technology for the extraction of flax lignans, which has the characteristics of simple operation and low equipment requirements, but also has the disadvantages of large consumption of organic solvents, long extraction time, and low yield.
  • the purpose of the present invention is to provide a subcritical composite solvent extraction and separation method for multiple components in flaxseed meal.
  • the method of the present invention obtains two natural extracts of flaxseed lignan and oil by optimizing the composite extraction solvent and extraction conditions. After the extraction, the waste residue continues to extract the linseed polysaccharide (gum).
  • the unsaturated fatty acid content of the linseed oil extracted by this method is as high as 90%, and its fatty acid content and glyceride composition are almost consistent with the refined linseed oil.
  • a subcritical composite solvent extraction and separation method for multiple components in linseed meal comprising the following steps:
  • the extractant is composed of n-butane and absolute ethanol in a volume ratio of 1:1; the addition of absolute ethanol adjusts the polarity of the composite solvent, facilitates the stripping of partial polar phenolic substances in flax meal, thereby improving the extraction efficiency.
  • the subcritical extraction is preferably carried out at 69°C for 2 hours;
  • the obtained lignan crude extract is rotary evaporated to remove residual extractant, centrifuged, and the obtained supernatant contains lignan polymer; the supernatant is subjected to alkali treatment, acid neutralization, macroporous adsorption resin chromatography, silica gel column Chromatography to obtain lignans with a purity > 90%;
  • alkali treatment it is preferred to add 0.1M sodium hydroxide solution for alkali hydrolysis at room temperature for at least 24 hours;
  • the acid neutralization preferably adds acetic acid until the pH of the system is 7;
  • the macroporous adsorption resin chromatography is preferably AB-8 macroporous resin adsorption, preferably eluting with 70% V/V ethanol solution to obtain an eluent containing lignans;
  • the extraction is preferably performed at 70°C for 3-5h;
  • step (3) taking the flaxseed meal used in step (1), and extracting it with hot water to obtain flaxseed gum;
  • the hot water extraction is performed under the conditions of 70° C., 2 hours, 400 rpm, and a solid-to-liquid ratio of 1:25.
  • the present invention has the following advantages and effects:
  • the subcritical composite solvent used in the extraction process of the present invention can replace a large amount of organic reagents used in the traditional extraction, and has the characteristics of good solvent effect, green recyclability, and small environmental burden, which can reduce the impact of organic reagents on the operation in the process. Risk of harm to people and the natural environment. And the subcritical extraction in the present invention has lower requirements on operation and professionals.
  • Flax lignans extracted by the present invention i.e. secoisolaricis resinol diglucoside (SDG)
  • SDG secoisolaricis resinol diglucoside
  • the linseed oil extracted by the present invention is different from traditional organic solvent extraction. Under subcritical conditions, the composite solvent will change its physical properties such as viscosity, diffusion coefficient and polarity with the increase of temperature and pressure, and its solubility can be improved. Compared with organic solvents, while ensuring the quality of oil, it avoids the problems of excessive use and residue of organic solvents in the extraction process.
  • the linseed gum can also be obtained by hot water extraction.
  • the source of the material is natural, the operation process is simple, the condition is mild, no large-scale equipment is needed, the cost is low, and the obtained linseed gum has the same apparent properties as those obtained in the prior art.
  • Fig. 1 is a technical roadmap of the extraction separation, purification and analysis method of the present invention.
  • Figure 2 is a graph showing the optimization results of the subcritical composite solvent ratio for extracting flax lignans.
  • Fig. 3 is a graph showing the optimization results of extraction temperature for subcritical extraction of flax lignans.
  • Fig. 4 is a comparison and optimization result diagram of extraction time for subcritical extraction of lignans.
  • Fig. 5 is a liquid chromatogram of SDG content after subcritical extraction of lignans by alkaline hydrolysis.
  • Figure 6 is a liquid chromatogram of subcritically extracted lignans purified by basic alumina and AB-8 macroporous adsorption resin.
  • Fig. 7 is a liquid chromatogram of subcritically extracted lignans purified by silica gel column chromatography.
  • Fig. 8 is a gas chromatogram of fatty acid composition of subcritically extracted linseed oil.
  • Figure 9 is a gas chromatogram of the glyceride composition of subcritically extracted linseed oil.
  • total phenol content determination adopts Folin's phenol method to measure, specifically as follows:
  • Gallic acid was used as the standard control. Weigh 20mg of gallic acid standard substance, add distilled water to dissolve it, transfer it to a 100mL volumetric flask, add distilled water to the lowest point of the concave liquid surface on the scale line, and shake well to obtain a gallic acid stock solution.
  • the analysis method of fatty acid composition and glyceride composition in linseed oil is as follows:
  • Fatty acid composition analysis is carried out first by methyl esterification reaction, take a drop of linseed oil into a 50mL round bottom flask, add 2mL 0.5M KOH methanol solution, and reflux in a water bath at 70°C for 10min. Add 3mL of BF 3 methanol solution after the reaction is finished and cool, then reflux in a water bath at 70°C for 5min, add 2-3mL of n-hexane after cooling, then add 2mL of saturated NaCl, react for 1min, then add an appropriate amount of anhydrous sodium sulfate , take the upper layer of n-hexane, pass it through a 0.45 ⁇ m membrane, inject it into the sample bottle, and then perform gas phase analysis.
  • Glyceride composition analysis is to dissolve 1 drop of oil in 2mL of n-hexane, inject it into a sample bottle through a 0.45 ⁇ m membrane, and then perform gas phase analysis.
  • Determination of total sugar content in flax meal by phenol-sulfuric acid method Accurately weigh 10mg of standard glucose into a 100mL volumetric flask, add distilled water to the mark, absorb 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6mL respectively, make up to 2.0mL with water, then add 6% phenol 1.0 mL and 5.0mL of concentrated sulfuric acid, let stand for 10min, shake well, measure the absorbance at 490nm after standing at room temperature for 20min, use 2.0mL water as a blank according to the same color development operation, the ordinate is the absorbance, and the abscissa is the glucose content ( ⁇ g). Linear regression was used to obtain the equation of the standard curve.
  • the content of reducing sugar in flax meal was determined by 3,5-dinitrosalicylic acid method. Accurately weigh 50mg of standard glucose into a 100mL volumetric flask, add distilled water to the mark, absorb 0.1, 0.2, 0.3, 0.4 and 0.5mL respectively, make up to 0.5mL with water, then add 0.5mL DNS, heat in a boiling water bath for 5min, Cool to room temperature immediately after taking it out, add 4mL of distilled water to each tube, mix well, measure the absorbance at 540nm, use 0.5mL of distilled water as a blank according to the same color development operation, the ordinate is the absorbance, the abscissa is the glucose content ( ⁇ g), and perform linear regression to obtain a standard curve.
  • Polysaccharide content total sugar content - reducing sugar content
  • a subcritical composite solvent extraction and separation method for various components of linseed meal comprises the following steps:
  • the upper layer of the extract is the crude lignan extract, which is concentrated by rotary evaporation at 40° C. to remove ethanol and a small amount of residual n-butane. After concentrating to a certain volume, centrifuge at 10,000 rpm for 10 minutes, and take the supernatant to obtain the lignan polymer.
  • the lower layer of the extract is linseed crude oil.
  • step (2) Take the crude linseed oil obtained in step (1), add n-hexane for liquid separation and extraction, collect the supernatant, concentrate by rotary evaporation until the volume remains constant, and obtain linseed oil.
  • fatty acid composition is palmitic acid 16:0 (7.63%), stearic acid 18:0 (3.27%), oleic acid 18: 1 (13.75%), linoleic acid 18:2 (13.75%) and linolenic acid 18:3 (56.32%), the total content of unsaturated fatty acids is as high as 90%.
  • the composition of glyceride was fatty acid (3.46%), diglyceride (2.60%) and triglyceride (93.83%), all of which had no significant difference with the composition of commercial flaxseed oil. Therefore, subcritical composite solvent extraction of linseed meal has a high yield of residual oil, good quality, and no organic solvent residue. It is a green and efficient way of linseed oil extraction.
  • step (3) The linseed meal powder in the subcritical extraction kettle in step (1) is taken out, and air-dried or vacuum-dried to obtain constant-weight linseed meal powder.
  • the linseed gum was extracted with deionized water, and the linseed meal was extracted with water under the conditions of 70°C, 3h, 400rpm, and a material ratio of 1:25.
  • the content of polysaccharide in water-extracted linseed gum was 18.19%.
  • step (1) Take the supernatant containing lignan polymer obtained in step (1), add 5 times the volume of 0.1M sodium hydroxide solution, perform alkaline hydrolysis at room temperature for 24 hours, add glacial acetic acid to neutralize to a pH value of 7 , it can be clearly observed that when the pH value is close to 7.0, the orange-yellow clear liquid briefly turns into a lemon-yellow turbid state, and the neutralized liquid is condensed by vacuum evaporation and freeze-dried to obtain the crude product of flax lignan (SDG).
  • SDG crude product of flax lignan
  • Glacial acetic acid neutralization refers to placing the sample solution after alkali hydrolysis on a magnetic stirrer, adding AR grade glacial acetic acid in small amounts and multiple times while stirring, and monitoring in real time with precision pH test paper or pH meter.
  • the content of SDG in the neutralized liquid was determined by HPLC, and the extraction rate was 6.99% (Fig. 5).
  • Redissolve 5g of the crude SDG sample in 1000mL of 10% ethanol solution then pass through 200-300g of basic alumina to remove the organic acid, filter 2-3 times to obtain the filtrate, add AB-8 macroporous adsorption resin to absorb 4 -10h, during which a glass rod can be used to stir intermittently. After the adsorption is completed, obtain a macroporous resin by suction filtration, rinse the resin surface with deionized water several times, then add 1000 mL of 70% ethanol solution to soak the resin for 4-10 hours, and stir intermittently with a glass rod during this period to obtain ethanol that resolves SDG solution.
  • HPLC monitoring AB-8 macroporous adsorption resin treatment obtained SDG with a sample purity of about 84% ( FIG. 6 ).
  • the obtained ethanol solution was concentrated to dryness by rotary evaporation at 40°C.
  • the converted flax lignans SDG content in flax meal was 4.19mg/g.

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Abstract

本发明公开了亚麻籽粕中多种成分的亚临界复合溶剂萃取分离方法，包括以下步骤：取亚麻籽粕，加入萃取剂，进行亚临界萃取，一步得到亚麻木酚素粗提液和亚麻籽粗油；所述的萃取剂由正丁烷和无水乙醇按体积比1:1组成；亚麻籽粗油加入正己烷进行分液萃取，旋转蒸发后可得亚麻籽油；取用后的亚麻籽粕，热水浸提获得亚麻籽胶。本发明萃取过程中使用的亚临界复合溶剂，可替代传统萃取中使用的大量有机试剂，具有溶剂效果好，绿色可回收，环境负担小等特性，可减少有机试剂在过程中对操作人员和自然环境造成的危害风险。且本发明中亚临界萃取对操作和专业人员要求更低，在减少操作单元的同时获得多种高附加值成分，资源利用最大化，降本增效。

Description

亚麻籽粕中多种成分的亚临界复合溶剂萃取分离方法 技术领域
本发明属于植物有效成分的萃取分离领域，具体涉及亚麻籽粕中多种活性成分的亚临界复合溶剂萃取分离方法。
背景技术
亚麻籽油富含α-亚麻酸、V E等营养物质。亚麻籽经压榨后所得饼粕仍含有残油，以及木酚素、多糖(胶)和蛋白等活性成分。作为亚麻籽油加工的主要副产物，亚麻籽饼粕通常仅被用作动物饲料、肥料或者废料处理，资源未得到充分利用。
目前，我国的亚麻籽产品主要以亚麻籽油为主，产品单一且同质化严重，亚麻籽功能性食品配料的开发逐渐成为热点。油脂的制取工艺有压榨法、溶剂萃取法和超临界CO 2萃取法，其中压榨法得率低，处理量有限，往往采取溶剂萃取法弥补缺陷，但该法所得油脂品质有差异，且有溶剂残留风险。新兴的超临界CO 2萃取法得率较高，品质优良，但工业化生产成本较高，需要专业化人士操作。
除亚麻籽油外，亚麻木酚素等其他高附加值活性成分的高效绿色萃取研究较少。目前，亚麻木酚素存在于亚麻籽壳中，其萃取工艺主要分为有机溶剂萃取法、微波辅助萃取法和超声辅助萃取法。有机溶剂法是目前亚麻木酚素萃取的主要技术，具有操作简捷、设备要求低等特点，但也存在有机溶剂消耗量大，萃取时间长，得率较低等缺点。
技术问题
在溶剂萃取法的基础上采用微波或超声波辅助可以大大缩短萃取时间，且在一定程度上可提高亚麻木酚素的萃取率，但对设备和操作人员有一定要求，离产业化还有距离。
迄今为止，以亚麻籽粕为原料，一步同时萃取获得亚麻木酚素和亚麻籽油，进而获得亚麻籽胶的高效综合利用技术鲜有报道。
技术解决方案
本发明的目的在于提供亚麻籽粕中多种成分的亚临界复合溶剂萃取分离方法，本发明方法通过对复合萃取溶剂和萃取条件的优化，同时得到亚麻籽木酚素和油两种天然萃取物，萃取后废渣继续进行亚麻多糖(胶)的萃取，该方法萃取的亚麻籽油不饱和脂肪酸含量高达90％，其脂肪酸含量和甘油酯组成与精炼亚麻籽油几乎一致。
本发明的目的通过下述技术方案实现：
亚麻籽粕中多种成分的亚临界复合溶剂萃取分离方法，包括以下步骤：
(1)取亚麻籽粕，加入萃取剂，进行亚临界萃取，萃取后萃取液上层为木酚素粗提物，萃取液下层为亚麻籽粗油；
所述的萃取剂由正丁烷和无水乙醇按体积比1:1组成；无水乙醇的加入调整了复合溶剂的极性，便于亚麻粕中偏极性酚类物质的溶出，从而提高萃取效率。
所述的亚临界萃取，优选在69℃下进行亚临界萃取2h；
所得到的木酚素粗提液，旋转蒸发除去残留萃取剂，离心，所得上清液含有木酚素聚合物；上清液进行碱处理、酸中和、大孔吸附树脂层析、硅胶柱层析，得到纯度＞90％的木酚素；
所述的碱处理，优选加入0.1M的氢氧化钠溶液室温下碱水解至少24h；
所述的酸中和优选加入醋酸至体系pH值为7；
所述的大孔吸附树脂层析，优选AB-8大孔树脂吸附，优选以70％V/V的乙醇溶液洗脱，得到含有木酚素的洗脱液；
所述的柱层析，是以二氯甲烷、二氯甲烷/甲醇/冰乙酸＝10:1:0.1(V/V/V)、二氯甲烷/甲醇/冰乙酸＝10:2:0.1(V/V/V)、二氯甲烷/甲醇＝10:2.5:0.1(V/V/V)依次洗脱，收集SDG组分的所有管液；
(2)取步骤(1)所得亚麻籽粗油，加入正己烷进行分液萃取，取上清液，旋蒸浓缩至体积不变后即为亚麻籽油，该方法萃取的亚麻籽油中不饱和脂肪酸含量高达90％；
所述的萃取优选在70℃下萃取3-5h；
(3)取步骤(1)用后的亚麻籽粕，热水浸提获得亚麻籽胶；
所述的热水浸提，是在70℃、2h、400rpm、料液比1:25的条件下水提。
有益效果
本发明相对于现有技术具有如下的优点及效果：
(1)本发明萃取过程中使用的亚临界复合溶剂，可替代传统萃取中使用的大量有机试剂，具有溶剂效果好，绿色可回收，环境负担小等特性，可减少有机试剂在过程中对操作人员和自然环境造成的危害风险。且本发明中亚临界萃取对操作和专业人员要求更低。
(2)本发明萃取所得亚麻木酚素，即开环异落叶松树脂酚二葡糖苷(SDG)，具有较好的抗氧化、抗骨质疏松等生物活性，纯化后纯度可达>90％，可进一步开发成为食品或保健品功能配料。相对于现有技术，本发明萃取在最大程度上规避了传统萃取过程中有机溶剂大量使用和残留的问题，在减少操作单元的同时获得了亚麻籽油，降本增效。
(3)本发明萃取所得亚麻籽油，区别于传统有机溶剂萃取，在亚临界条件下，复合溶剂随着温度和压力的增加会改变其粘度、扩散系数和极性等物理特性，溶解能力可比拟有机溶剂，从而在保证油脂品质的同时，规避了萃取过程中有机溶剂大量使用和残留等问题。
(4)本发明一步萃取出亚麻籽木酚素和油后，还可通过热水浸提法得到亚麻籽胶。材料来源天然，操作过程简单，条件温和，无需大型设备，成本低廉，得到的亚麻籽胶表观特性与现有技术所得无异。
附图说明
图1是本发明萃取分离、纯化和分析方法的技术路线图。
图2是萃取亚麻木酚素的亚临界复合溶剂配比优化结果图。
图3是亚临界萃取亚麻木酚素的萃取温度优化结果图。
图4是亚临界萃取木酚素的萃取时间比较优化结果图。
图5是亚临界萃取木酚素的碱水解后SDG含量的液相色谱图谱。
图6是亚临界萃取的木酚素经过碱性氧化铝和AB-8大孔吸附树脂纯化后的液相色谱图谱。
图7是亚临界萃取的木酚素经过硅胶柱层析纯化后的液相色谱图谱。
图8是亚临界萃取亚麻籽油的脂肪酸组成气相色谱图。
图9是亚临界萃取亚麻籽油的甘油酯组成气相色谱图。
本发明的实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
本发明中，总酚含量测定采用福林酚法测定，具体如下：
采用没食子酸作为标准品对照。称取没食子酸标准品20mg加蒸馏水溶解后转移至100mL容量瓶中，加蒸馏水水定容至刻度线凹液面最低处，摇匀，得没食子酸储备液。
精密量取上述储备液1.0mL、2.0mL、3.0mL、4.0mL、5.0mL，分别置于10mL容量瓶中，加水至刻度，摇匀，得到一系列不同浓度的没食子酸标准溶液。
1)空白溶液配制：将没食子酸标准溶液1.0mL分别移入试管内，并分别加入5.0mL福林酚试剂，摇匀。反应3～8分钟后，加入7.5％的Na 2CO 3溶液4.0mL，摇匀，室温下放置60min。
2)标准曲线绘制：以空白溶液为对照，在765nm波长下测定吸光度。以吸光度为纵坐标，浓度为横坐标，绘制回归曲线y＝5.983X+0.006，计算线性回归方差。
3)样品溶液分析：取样品溶液1.0mL，并加入福林酚试剂5.0mL,摇匀后于765nm波长处测定吸光度。实验重复多次，样品中的总酚含量(Total phenol content，TPC)以每克干重的的毫克没食子酸当量(mg GAE/g dw)表示。
本发明中，亚麻籽油中脂肪酸组成和甘油酯组成的分析方法如下：
脂肪酸组成分析是先进行甲酯化反应，取一滴亚麻籽油到50mL圆底烧瓶中，加入2mL 0.5M KOH甲醇溶液，在70℃的条件下水浴回流10min。待反应结束冷却后加入3mL的BF 3甲醇溶液，再在70℃的条件下水浴回流5min，冷却后先加入2-3mL正己烷，再加入2mL饱和NaCl，反应1min后，加入适量无水硫酸钠，取上层正己烷，过0.45μm的膜打进进样瓶里，然后进行气相分析。
甘油酯组成分析是将1滴油溶解在2mL的正己烷中，过0.45μm的膜打进进样瓶里，然后进行气相分析。
利用苯酚-硫酸法测定亚麻粕中总糖的含量。准确称取标准葡萄糖10mg于 100mL容量瓶中，加蒸馏水至刻度，分别吸取0.2、0.4、0.6、0.8、1.0、1.2、1.4及1.6mL，各以水补至2.0mL，然后加入6％苯酚1.0mL及浓硫酸5.0mL，静置10min，摇匀，室温放置20min以后于490nm测吸光度，以2.0mL水按同样显色操作作为空白，纵坐标作为吸光度，横坐标为葡萄糖含量(μg)，进行线性回归得到标准曲线方程。
采用3,5-二硝基水杨酸法测定亚麻粕中还原糖的含量。准确称取标准葡萄糖50mg于100mL容量瓶中，加蒸馏水至刻度，分别吸取0.1、0.2、0.3、0.4及0.5mL，各以水补至0.5mL，然后加入0.5mL DNS，沸水浴中加热5min，取出后立即冷却至室温，各管加蒸馏水4mL，充分混匀，540nm测定吸光度，以0.5mL蒸馏水按同样显色操作作为空白，纵坐标为吸光度，横坐标为葡萄糖含量(μg)，进行线性回归得到标准曲线。
多糖含量的计算：
多糖含量＝总糖含量-还原糖含量
多糖的萃取得率＝(m l-m 2)/m×100％
式中：m 1-亚麻总糖的质量，g；m 2-亚麻还原糖的质量，g；m-亚麻籽粕粉的质量，g。
实施例
亚麻籽粕多种成分的亚临界复合溶剂萃取分离方法，包括以下步骤：
(1)称取10.0g研磨好的亚麻籽粕粉放置在亚临界萃取釜中的萃取袋里，加入200mL的正丁烷，进行萃取条件的单因素试验，探究不同复合溶剂(正丁烷与无水乙醇)配比(图2)、萃取温度(图3)、萃取时间(图4)对总酚含量的影响，根据单因素最优值结果进行响应面设计实验，得到亚临界萃取最优条件为复合溶剂配比1:1(正丁烷与无水乙醇)，萃取温度69℃，萃取时间2h，所得样品总酚含量为为22.76mg GAE/g。
萃取后萃取液上层为木酚素粗提物，在40℃下进行旋转蒸发浓缩除去乙醇和微量残留的正丁烷。浓缩到一定体积后，转速为10000rpm条件下离心10min，取上清液后得木酚素聚合物。
萃取液下层为亚麻籽粗油。
(2)取步骤(1)所得亚麻籽粗油，加入正己烷分液萃取，收集上清液，旋蒸浓缩至体积不变即为亚麻籽油。
所得亚麻籽油脂肪酸和甘油酯组成的气相分析结果如图8和图9所示，脂肪酸组成为棕榈酸16:0(7.63％)、硬脂酸18:0(3.27％)、油酸18:1(13.75％)、亚油酸18:2(13.75％)和亚麻酸18:3(56.32％)，不饱和脂肪酸总含量高达90％。甘油酯组成为脂肪酸(3.46％)、甘油二酯(2.60％)和甘油三酯(93.83％)，均与市售亚麻籽油组成无显著性差异。因此，亚临界复合溶剂萃取亚麻粕中的残油得率较高，品质良好，无有机溶剂残留，是一种绿色高效的亚麻籽油萃取的方式。
(3)将步骤(1)中亚临界萃取釜中的亚麻籽粕粉取出，采用风干或真空干燥的方式，得到恒重的亚麻粕粉。用去离子水浸提亚麻籽胶，亚麻籽粕在70℃、3h、400rpm、物料比1:25的条件下水提亚麻籽胶。水提亚麻籽胶中多糖含量为18.19％。
(4)取步骤(1)得到的含有木酚素聚合物的上清液，加入5倍体积的0.1M的氢氧化钠溶液，室温下碱水解24h，加入冰醋酸中和至pH值为7，可以明显观察到pH值接近7.0时，橙黄色的清澈液体短暂地变成柠檬黄的浑浊状态，将中和液体真空蒸发凝缩和冷冻干燥得到亚麻木酚素(SDG)粗品。
冰醋酸中和是指将碱水解后的样品液放置于磁力搅拌器上，一边搅拌一边少量多次加入AR级冰醋酸，并用精密pH试纸或pH计实时监测。
HPLC测定中和液体中SDG的含量，萃取率为6.99％(图5)。
将5g SDG粗品样品重溶于1000mL的10％的乙醇溶液中，然后先通过200-300g碱性氧化铝除去有机酸，抽滤2-3次得到滤液，加入AB-8大孔吸附树脂吸附4-10h，期间可用玻璃棒间歇性地搅拌。吸附结束后抽滤得到大孔树脂，用去离子水多次冲洗树脂表面，再加入1000mL的70％的乙醇溶液浸泡树脂4-10h，期间亦用玻璃棒间歇性地搅拌得到解析出SDG的乙醇溶液。
HPLC监测AB-8大孔吸附树脂处理得到样品纯度约为84％的SDG(图6)。
将得到的乙醇溶液在40℃的条件下旋蒸浓缩至干燥。在烧瓶中加入SDG质量约3-5倍质量的硅胶粉进行硅胶柱层析，分别以二氯甲烷：甲醇：冰乙酸＝10:1： 0.1、二氯甲烷：甲醇：冰乙酸＝10:2:0.1和二氯甲烷：甲醇：冰乙酸＝10:2.5:0.1的体积比进行梯度洗脱。
所述的柱层析，以SDG标准品为对照，用内径为0.3mm的玻璃点样毛细管吸取洗脱液，将洗脱液用硅胶薄层层析板进行层析分离，展开剂为二氯甲烷:甲醇:冰乙酸＝10:3:0.1，层析完成后，在紫外灯254nm下进行紫外显色分析。根据TLC法分析结果，逐步调整洗脱剂极性，分别以二氯甲烷、二氯甲烷/甲醇/冰乙酸＝10:1:0.1(V/V/V)、二氯甲烷/甲醇/冰乙酸＝10:2:0.1(V/V/V)、二氯甲烷/甲醇＝10:2.5:0.1(V/V/V)进行梯度洗脱，40mL/管收集洗脱液，直至SDG组分洗脱完全。收集SDG组分的所有管液，减压浓缩干燥，利用真空泵除去残留溶剂，之后利用高效液相色谱法鉴定纯度。
将84％的SDG经过硅胶柱层析处理，得到纯度约为94％的SDG(图7)。
折算亚麻粕中亚麻木酚素SDG含量为4.19mg/g。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 亚麻籽粕中多种成分的亚临界复合溶剂萃取分离方法，其特征在于包括以下步骤：
    (1)取亚麻籽粕，加入萃取剂，进行亚临界萃取，萃取后萃取液上层为木酚素粗提物，萃取液下层为亚麻籽粗油；
    所述的萃取剂由正丁烷和无水乙醇按体积比1:1组成；
    (2)取步骤(1)所得亚麻籽粗油，加入正己烷进行分液萃取，取上清液，旋蒸浓缩至体积不变后即为亚麻籽油；
    (3)取步骤(1)用后的亚麻籽粕，热水浸提获得亚麻籽胶。
  2. 根据权利要求1所述的萃取分离方法，其特征在于：步骤(1)所述的亚临界萃取，是在69℃下进行亚临界萃取2h。
  3. 根据权利要求1所述的萃取分离方法，其特征在于：所得到的木酚素粗提液，旋转蒸发除去残留萃取剂，离心，所得上清液含有木酚素聚合物；上清液进行碱处理、酸中和、大孔吸附树脂层析、硅胶柱层析，得到木酚素。
  4. 根据权利要求3所述的萃取分离方法，其特征在于：所述的硅胶柱层析，是以二氯甲烷、二氯甲烷/甲醇/冰乙酸＝10:1:0.1(V/V/V)、二氯甲烷/甲醇/冰乙酸＝10:2:0.1(V/V/V)、二氯甲烷/甲醇＝10:2.5:0.1(V/V/V)依次洗脱，收集SDG组分的所有管液。
  5. 根据权利要求3所述的萃取分离方法，其特征在于：所述的碱处理，是加入0.1M的氢氧化钠溶液室温下碱水解至少24h。
  6. 根据权利要求3所述的萃取分离方法，其特征在于：所述的酸中和是加入醋酸至体系pH值为7。
  7. 根据权利要求3所述的萃取分离方法，其特征在于：所述的大孔吸附树脂层析，是采用AB-8大孔树脂吸附。
  8. 根据权利要求7所述的萃取分离方法，其特征在于：所述的大孔吸附树脂层析，以70％V/V的乙醇溶液洗脱，得到含有木酚素的洗脱液。
  9. 根据权利要求1所述的萃取分离方法，其特征在于：步骤(2)所述的分液萃取是在70℃下萃取3-5h。
  10. 根据权利要求1所述的萃取分离方法，其特征在于：步骤(3)所述的热水浸提，是在70℃、2h、400rpm、料液比1:25的条件下水提。
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