CN114073864A - Method for synchronously extracting and separating multiple components in raw material by four-liquid-phase system - Google Patents
Method for synchronously extracting and separating multiple components in raw material by four-liquid-phase system Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and discloses a method for synchronously extracting and separating multiple components in raw materials by a four-liquid-phase system, which is characterized in that the raw materials containing various components with different polarities are mixed with water, soluble salt and a hydrophilic solvent to prepare the four-liquid-phase system; and centrifuging and/or standing the four-liquid-phase system to form four liquid phases, respectively extracting each component in the raw materials into each layer of the strong hydrophobic layer, the weak hydrophilic layer and the strong hydrophilic layer according to the polarity difference, and distributing insoluble residues between the interfaces of the phases. The method can effectively perform simultaneous extraction and separation on crude extracts with complex components, has the advantages of low energy consumption, simple separation steps and the like, and solves the problems of complex operation, easy product loss, high production cost and the like of the current multi-component product synchronous extraction and separation.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for synchronously extracting and separating multiple components in a complex raw material by a four-liquid-phase system.
Background
In the fields of food, medicine, traditional Chinese medicine, cosmetics and the like, the extraction and separation of substances with economic value are often related, and the components can be roughly divided into substances with strong hydrophobicity such as fatty oil and the like according to the polarity; weakly hydrophobic substances such as organic acids; weakly hydrophilic substances such as flavonoids; polysaccharides and other strongly hydrophilic substances. Most of the substances are difficult to extract and separate synchronously, most processes are concentrated on extracting one to two main components, and a plurality of valuable components are lost and wasted in the extraction, so that not only is a huge loss caused, but also a huge pressure is brought to the environmental pollution. Therefore, the method synchronously extracts and separates each component from the complex components for comprehensive utilization, is a difficult point and a hotspot of the current research, and has great research significance and application value. For example, Antarctic krill oil contains various substances with different hydrophilicities and hydrophobicities, including astaxanthin, phospholipid, triglyceride, adenosine, protein, saccharides and the like, and is often in the form of a mixture in the extraction process, all components are mixed with the krill oil, but most of the current applications can only extract part of astaxanthin and phospholipid, and more triglyceride is mixed, so that the product quality is seriously influenced, and the comprehensive application of the product is restricted.
For obtaining target components from complex components, the traditional oil-water system extraction, solvent extraction, supercritical fluid extraction, three-liquid phase extraction and the like are adopted, the solvent extraction is mostly used for extraction and separation, and the traditional separation and purification means needs extraction, extraction and repeated chromatographic separation for many times to obtain high-purity compounds. In the prior patent, for the extraction of polyphenols such as tea saponin, flavone and the like in tea seed cake, organic solvent petroleum ether is added for extraction, solid-liquid separation and drying to obtain degreased tea seed oil cake, then solvents such as ethanol and the like are added, macroporous adsorption resin is used for extraction and separation of tea saponin and polyphenol (CN 102993329A), solvent extraction and resin adsorption are repeatedly used, and the process is complex and time-consuming. The lipase catalyzes and hydrolyzes the grease in a traditional oil-water system, the hydrolysis product glycerol and the catalyst lipase are in the same phase, the hydrolysis behavior of the enzyme is inhibited by high-concentration glycerol, and the dispersibility of the grease is too small and the particle size of the emulsion is too large in the oil-water system (Chinese grease, 2013,38(07): 56-59). The solvent extraction method is mainly used industrially, and generally, single components are extracted, different substances are extracted step by step, and the problems of low extraction purity, easy loss, incomplete separation, sample denaturation and the like exist. The most common solvent extraction method is used for extracting the oil and the astaxanthin in the antarctic krill at present, but the problems of large solvent consumption, difficult recovery and treatment, long extraction time and the like exist. The extraction of krill oil and astaxanthin by the supercritical fluid method can obtain relatively pure target substances, but extraction needs to be respectively arranged, extraction of each substance needs to be carried out step by step, and the supercritical fluid method has the problems of high requirement on equipment, high extraction cost and the like, is only suitable for laboratory scale (food industry science and technology, 2021,42(12): 362-368) and cannot be put into industrial production. Although an oil-water system or a three-liquid system can separate hydrophilic and hydrophobic substances, it cannot separate lipophilic substances having both hydrophobicity and hydrophobicity. Therefore, how to synchronously extract and separate complex mixtures in the production process so as to realize deep utilization of resources and obtain high-yield and high-purity active substances is still the focus of current research.
Disclosure of Invention
The invention aims to provide a method for synchronously extracting and separating multiple components of a complex raw material based on a four-liquid-phase system, aiming at the problems that the steps of simultaneously extracting and separating the multiple components of an active component are complicated, time-consuming, easy to cause loss and the like.
In recent research in the laboratory, it was found that when a certain content of surfactant is reached in a mixture of biolipid and a hydrophobic organic solvent, a four-liquid-phase system can be formed by adding water, soluble salt and a hydrophilic solvent in a certain proportion, and multi-components can be synchronously extracted and separated into four phases according to the polarity difference of components, so that not only can substances with larger polarity difference be separated, but also the problem that hydrophobic substances with similar polarity cannot be simultaneously separated can be improved, for example, two hydrophobic substances of phospholipid, triglyceride and astaxanthin in antarctic krill oil can be synchronously separated. And simultaneously, the protein and the saccharide can be separated, the extraction and separation process is simple, the time consumption is short, the interference among the separated components is small, the toxicity is small, the green is realized, and an effective method is provided for the separation of multiple components with small polarity difference.
The purpose of the invention is realized by the following technical scheme:
a method for synchronously extracting and separating multiple components in a raw material by a four-liquid-phase system comprises the following steps:
(1) mixing raw materials containing various components with different polarities with water, soluble salt and a hydrophilic solvent to prepare a four-liquid-phase system; the system contains hydrophobic substances and surfactants, wherein the surfactants are amphoteric surfactants and/or lipophilic nonionic surfactants;
(2) and centrifuging and/or standing the prepared four-liquid-phase system to form four liquid phases, respectively extracting each component in the raw materials into each layer of the strong hydrophobic layer, the weak hydrophilic layer and the strong hydrophilic layer according to the polarity difference, and distributing insoluble residues between phase interfaces.
When the raw materials of the system contain hydrophobic substances such as vegetable oil (such as soybean oil) and lipophilic nonionic surfactants such as monoglyceride laurate and monoglyceride stearate and/or amphoteric surfactants such as phospholipid and phosphatidylcholine, the system can form four phases without adding hydrophobic substances and surfactants.
Preferably, a hydrophobic substance is further added into the system in the step (1), and the mass volume ratio of the raw material to the hydrophobic substance is 0.1-4.0.
Preferably, the hydrophobic substance is one or more of isooctane, n-hexane, ethyl acetate, chloroform, triglyceride, diglyceride, monoglyceride, astaxanthin, phospholipid, vitamin a, tocopherol, squalene, vitamin D, sterol (cholesterol, campesterol, stigmasterol), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), oleic acid, and linoleic acid.
Preferably, a surfactant is further added into the system in the step (1), and the mass ratio of the raw material to the surfactant is 0.1-3.0; the surfactant is one or more of phospholipid, phosphatidylcholine, monoglyceride laurate and monoglyceride stearate.
Preferably, the hydrophilic solvent in step (1) is methanol, ethanol, isopropanol, PEG400, PEG600, [ BMIM ]]BF4、[BMIM]PF6One or a combination of several of them.
Preferably, the soluble salt in step (1) is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, sodium sulfate, sodium carbonate, ammonium sulfate and potassium chloride.
Preferably, the mass ratio of the water, the soluble salt, the hydrophilic solvent and the raw materials in the step (2) is (0.25-0.70): (0.13-0.60): (0.15-0.60): (0.3-1.0).
Preferably, the raw material in the step (1) is a pretreated raw material liquid; the mixing conditions in the step (1) are as follows: the temperature is 10-80 ℃, and the rotating speed is 100-1000 rpm.
Preferably, the pretreatment is one or more than two of hydrolysis, enzymolysis, ultrasonic crushing, homogenization and leaching.
Preferably, the raw materials in step (1) comprise antarctic krill oil, fish oil, lard, beef tallow, soybean oil, camellia oil, olive oil, walnut oil or other biolipid substances, tea seed cake powder, cordyceps sinensis powder and the like.
The surfactant and the hydrophobic substance in the system of the invention redistribute after being mixed with the hydrophilic solvent and the salt solution, thus providing possibility for forming two hydrophobic phases with different strengths. The strong hydrophobic phase, the weak hydrophilic phase and the strong hydrophilic phase which are separated in the step (2) can be respectively taken out and mixed according to different volume ratios to obtain a new system, and the yield can be improved.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for synchronously extracting and separating multi-component products based on a four-liquid-phase system, which solves the problems that the steps of simultaneously extracting and separating multi-component active ingredients are complicated, time-consuming, easy to cause loss and the like. The concrete points are as follows: (1) overcomes the difficulty that the prior active ingredients with various polarities can not be separated simultaneously, and particularly can be synchronously separated for a mixture simultaneously containing various fat-soluble ingredients. (2) The method can simultaneously realize extraction and separation of multiple components, has the advantages of low energy consumption, simple separation steps and the like, and solves the problems of complex operation, easy product loss, high production cost and the like of the conventional multi-component product synchronous extraction and separation. (3) Can be applied to the synchronous extraction and separation of multi-component active ingredients extracted in solvents with different polarities, including water, hydrophilic solvents or grease.
Drawings
FIG. 1 is a phase diagram of the system of example 13; FIG. 1 (a) shows a two-phase system, FIG. 1 (b) shows a three-phase system, and FIG. 1 (c) shows a four-phase system.
FIG. 2 shows K in example 62HPO4A phase diagram of a four-liquid phase system of ethanol-n-hexane-antarctic krill oil (IV: the system can form four phases under the condition of the ratio of the saline to the alcohol inside the diagram, and III, II: the system can only form three phases or two phases under the condition of the ratio of the saline to the alcohol outside the diagram).
FIG. 3 shows Na in example 122SO4-PEG 600-monoglyceride laurate-soybean oil four-liquid phase system (IV: the system can form four phases under the condition of the ratio of the salt and the alcohol in the inner part of the graph, and III, II: the system can only form three phases or two phases under the condition of the ratio of the salt and the alcohol in the outer part of the graph).
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.
Example 1
Step (1): and uniformly stirring the crude animal oil mixed with the triglyceride and the diglyceride and the isooctane according to the mass volume ratio w/v =1:9 to obtain a crude oil solution.
Weighing 11.34g of water, adding 3.42g of sodium carbonate to prepare an inorganic salt solution, adding 3.24g of anhydrous ethanol, adding 9g of the crude oil solution, fully shaking and shaking uniformly, centrifuging for 3min at 9000r/min to form a four-liquid-phase system, wherein a strong hydrophobic phase (phase I), a weak hydrophobic phase (phase II), a weak hydrophilic phase (phase III) and a strong hydrophilic phase (phase IV) are respectively arranged from top to bottom, taking out in layers, respectively measuring the contents of Triglyceride (TAG), diglyceride (Sn-1, 2-DAG), protein and total sugar to obtain triglyceride and diglyceride distributed in hydrophobic phases, wherein 93.5% of triglyceride is distributed in the strong hydrophobic phase, the selectivity coefficient of Sn-1 and 2-DAG in the weak hydrophobic phase relative to the TAG in the strong hydrophobic phase reaches 9.02, the TAG and Sn-1,2-DAG can be well separated, the protein and the protein, The total sugars partition in the hydrophilic phase, with 88.2% of the protein partitioning in the less hydrophilic phase (phase III), the protein selectivity coefficient for the total sugars in the less hydrophilic phase versus the more hydrophilic phase being 22.9, the insolubles remaining in the crude oil solution between the less hydrophilic phase (phase III) and the more hydrophilic phase (phase IV), and 75.3% of the sugars partitioning in the more hydrophilic phase (phase IV), the sugars partitioning coefficient for the less hydrophilic phase versus the more hydrophilic phase being 0.62.
Example 2
Step (1): and uniformly stirring the crude animal oil mixed with the triglyceride and the fatty acid and the isooctane according to the mass volume ratio w/v =1:9 to obtain a crude oil solution.
Weighing 11.7g of water, adding 3.42g of sodium carbonate to prepare an inorganic salt solution, adding 2.88g of absolute ethyl alcohol, adding 9g of the crude oil solution, fully shaking and shaking uniformly, centrifuging for 3min at 9000r/min to form a four-liquid-phase system, wherein a strong hydrophobic phase, a weak hydrophilic phase and a strong hydrophilic phase are respectively arranged from top to bottom, taking out the four-liquid-phase system in a layering manner, and respectively measuring the contents of Triglyceride (TAG), fatty acid (FFA), protein and total sugar, wherein 92.2 percent of TAG is distributed in the strong hydrophobic phase, the selectivity coefficient of FFA in the TAG in the strong hydrophobic phase relative to the FFA in the weak hydrophobic phase reaches 260, the TAG and the FFA can be well separated, the protein and the total sugar are distributed in the hydrophilic phase, wherein 87.1 percent of the protein is distributed in the weak hydrophilic phase (III phase), the selectivity coefficient of the total sugar in the weak hydrophilic phase relative to the strong hydrophilic phase in the weak hydrophilic phase is 22, and the residual insoluble phase (second hydrophilic phase) in the initial pressing liquid is distributed between the strong hydrophilic phase (IV phase), 76.3% of the saccharides partition in the strongly hydrophilic phase (phase IV) and the partition coefficient of the saccharides in the weakly hydrophilic phase relative to the strongly hydrophilic phase is 0.70.
Example 3
Step (1): uniformly stirring the initial pressed antarctic krill oil and isooctane according to the mass-volume ratio w/v =1:9, adding 0.25% of pepsin and 0.25% of cellulase, hydrolyzing for 4h at 50 ℃, and then inactivating in a water bath at 80-100 ℃ for 15min to obtain an antarctic krill oil solution.
Weighing 10.98g of water, adding 3.42g of sodium carbonate into the water to prepare an inorganic salt solution, adding 3.6g of absolute ethyl alcohol, adding 9g of the antarctic krill oil solution, shaking uniformly and fully, centrifuging for 3min at 9000r/min to form a four-liquid-phase system, wherein the four-liquid-phase system comprises a strong hydrophobic phase, a weak hydrophilic phase and a strong hydrophilic phase from top to bottom, and taking out the phases in a layered manner. Preparing 2ml of strong hydrophilic phase (phase IV) in the same proportion, mixing with the four phases respectively taken out, centrifuging (namely changing the volume ratio of each phase in the system), taking out each layer, and respectively measuring the contents of triglyceride, phospholipid, protein and saccharide to obtain triglyceride and phospholipid which are distributed in hydrophobic phase, wherein 88.6% of triglyceride is distributed in strong hydrophobic phase (phase I), the phospholipid selectivity coefficient of triglyceride in strong hydrophobic phase and weak hydrophobic phase is 38.7, 84.8% of phospholipid is distributed in weak hydrophobic phase (phase II), the distribution coefficient of phospholipid in strong hydrophobic phase and weak hydrophobic phase is 0.07, protein and saccharide are distributed in hydrophilic phase, wherein 84.12% of protein is distributed in weak hydrophilic phase (phase III), the selectivity coefficient of protein in weak hydrophilic phase and strong hydrophilic phase is 12.6, and insoluble substances remained in squeezed liquid is between weak hydrophilic phase (phase III) and strong hydrophilic phase (phase IV), 70.25% of the saccharides partition into the strongly hydrophilic phase (phase IV) and the partition coefficient of the saccharides in the weakly hydrophilic phase relative to the strongly hydrophilic phase is 0.77.
Example 4
Step (1): uniformly stirring the initial pressed antarctic krill oil and isooctane according to the mass-volume ratio w/v =1:9, adding 0.25% of pepsin and 0.25% of cellulase, hydrolyzing for 4h at 50 ℃, and then inactivating in a water bath at 80-100 ℃ for 15min to obtain an antarctic krill oil solution.
Step (2): weighing 12.06g of water, adding 2.34g of sodium carbonate to prepare an inorganic salt solution, adding 3.6g of absolute ethyl alcohol, adding 9g of the antarctic krill oil solution, shaking uniformly and fully, centrifuging at 9000r/min for 3min to form a four-liquid-phase system, wherein the four-liquid-phase system comprises a strong hydrophobic phase, a weak hydrophilic phase and a strong hydrophilic phase from top to bottom, and taking out the phases in a layered manner. Preparing 5ml of strong hydrophobic phase (phase I) and 7ml of strong hydrophilic phase (phase IV) in the same proportion, mixing with the above four phases, centrifuging (changing volume ratio of each phase in the system), taking out each layer, respectively measuring the contents of triglyceride, phospholipid, protein and saccharide to obtain triglyceride and phospholipid distributed in hydrophobic phase, wherein 74.6% of the triglycerides are distributed in the strongly hydrophobic phase (phase I), the distribution coefficient of the triglycerides in the strongly hydrophobic phase relative to the weakly hydrophobic phase is 1.22, 100% of the phospholipids are distributed in the weakly hydrophobic phase (phase II), the proteins and carbohydrates are distributed in the hydrophilic phase, wherein 81.79% of the protein partitions into the less hydrophilic phase (phase III), the protein partitions into the less hydrophilic phase relative to the more hydrophilic phase with a partition coefficient of 8.98, 73.8% of the carbohydrate partitions into the more hydrophilic phase (phase IV), and the carbohydrate partitions into the less hydrophilic phase relative to the more hydrophilic phase with a partition coefficient of 0.711.
Example 5
Step (1): uniformly mixing soybean lecithin, soybean oil and n-hexane according to the mass ratio of w/w/w =1:9:7.5 in a water bath at 45 ℃ (dissolving the soybean lecithin) and under magnetic stirring at 600rpm to obtain a soybean oil solution.
Step (2): taking 10g of tea seed cake powder, carrying out ultrasonic crushing for 30min at 750w by using 70% ethanol solution, carrying out water bath extraction for 2h at 60 ℃, centrifuging for 5min at 10000r/min, and taking supernatant to obtain tea seed cake extracting solution.
And (3): weighing 9g of ultrapure water, dissolving 2.7g of dipotassium hydrogen phosphate to obtain an inorganic salt solution, adding 6.3g of the camellia dreg extracting solution, adding 9g of the mixed soybean oil, uniformly stirring, centrifuging at 9000r/min for 3min to form a four-liquid-phase system, wherein a strong hydrophobic phase, a weak hydrophilic phase and a strong hydrophilic phase are respectively arranged from top to bottom, and taking out in layers. Preparing 5ml strong hydrophilic phase (IV phase) at the same ratio, mixing with the above four phases respectively, centrifuging, taking out each layer, respectively measuring the contents of triglyceride, phospholipid, total flavone and tea saponin to obtain phospholipid and triglyceride distributed in hydrophobic phase, wherein 55.1% of phospholipid is distributed in strong hydrophobic phase (phase I), selectivity coefficient of phospholipid in strong hydrophobic phase relative to weak hydrophobic phase is 2.6, 67.3% of triglyceride is distributed in weak hydrophobic phase (phase II), distribution coefficient of triglyceride in strong hydrophobic phase relative to weak hydrophobic phase is 0.25, flavone and tea saponin are distributed in hydrophilic phase, wherein 100% of total flavone is distributed in weak hydrophilic phase (phase III), residual insoluble substance in the crude extractive solution is distributed between phase III and phase IV, 80.1% of tea saponin is distributed in strong hydrophilic phase (phase IV), and distribution coefficient of tea saponin in weak hydrophilic phase relative to strong hydrophilic phase is 0.21.
Example 6
Step (1): mixing antarctic krill oil with n-hexane according to a mass-to-volume ratio w/v =1:9, stirring and uniformly mixing to obtain the antarctic krill oil solution.
Step (2): weighing 11.16g of water, adding 2.34g of dipotassium hydrogen phosphate to prepare an inorganic salt solution, adding 4.5g of absolute ethyl alcohol, adding 9g of the antarctic krill oil solution, shaking uniformly and fully, centrifuging for 3min at 9000r/min to form a four-liquid-phase system, wherein a strong hydrophobic phase, a weak hydrophilic phase and a strong hydrophilic phase are respectively arranged from top to bottom, taking out the layers, and respectively measuring the contents of astaxanthin, phospholipid and saccharides to obtain astaxanthin and phospholipid which are distributed in the hydrophobic phases, wherein 68.6% of astaxanthin is distributed in the strong hydrophobic phase (phase I), the selectivity coefficient of astaxanthin in the strong hydrophobic phase and the weak hydrophobic phase is 11.2, 94.1% of phospholipid is distributed in the weak hydrophobic phase (phase II), the distribution coefficient of phospholipid in the strong hydrophobic phase and the weak hydrophobic phase is 0.17, saccharides are distributed in the hydrophilic phase, 90.6% of saccharides are distributed in the weak hydrophilic phase (phase III), and the distribution coefficient of saccharides in the weak hydrophilic phase and the weak hydrophilic phase is 4.6, the insoluble extraction residue partitions at the interface between the weakly hydrophilic phase (phase III) and the strongly hydrophilic phase (phase IV).
Example 7
Step (1): mixing antarctic krill oil with isooctane according to the mass-volume ratio w/v =1:9, stirring and uniformly mixing to obtain the antarctic krill oil solution.
Step (2): weighing 11.7g of water, adding 2.7g of sodium carbonate to prepare an inorganic salt solution, adding 3.6g of absolute ethyl alcohol, adding 9g of the antarctic krill oil solution, shaking up the mixture by full shaking, centrifuging the mixture for 3min at 9000r/min to form a four-liquid-phase system, wherein, from top to bottom, the phase is a strong hydrophobic phase, a weak hydrophilic phase and a strong hydrophilic phase, after each layer is taken out, respectively measuring the contents of astaxanthin, phospholipid and saccharide to obtain astaxanthin and phospholipid which are distributed in hydrophobic phase, wherein 98.8% of the astaxanthin is distributed in the strongly hydrophobic phase (phase I), the distribution coefficient of astaxanthin in the strongly hydrophobic phase relative to the weakly hydrophobic phase is 39.7, 100% of the phospholipids is distributed in the weakly hydrophobic phase (phase II), the saccharides are distributed in the hydrophilic phase, 90.5% of the saccharides are distributed in the weakly hydrophilic phase (phase III), the distribution coefficient of the saccharides in the weakly hydrophilic phase relative to the strongly hydrophilic phase is 12.5, and the extraction insoluble residue is distributed at the interface between the weakly hydrophilic phase (phase III) and the strongly hydrophilic phase (phase IV).
Example 8
Step (1): uniformly mixing soybean lecithin, soybean oil and n-hexane according to the mass ratio of w/w/w =1:9:3 in a water bath at 50 ℃ (soybean lecithin is dissolved) and under magnetic stirring at 500rpm to obtain a soybean oil solution.
Step (2): taking 10g Cordyceps powder, ultrasonically crushing with 70ml water at 250w for 30min, extracting in water bath at 80 deg.C for 3h, centrifuging at 10000r/min for 10min, and collecting supernatant to obtain Cordyceps powder extractive solution.
And (3): weighing 10.98g of the cordyceps sinensis powder extracting solution, adding 3.42g of dipotassium phosphate, dissolving and uniformly mixing to obtain a salt solution, adding 3.6g of absolute ethyl alcohol, adding 9g of the mixed soybean oil solution, stirring uniformly, centrifuging for 3min at 9000r/min to form a four-liquid-phase system, wherein a strong hydrophobic phase, a weak hydrophilic phase, a weak hydrophobic phase and a strong hydrophilic phase are respectively arranged from top to bottom, taking out each layer, respectively measuring the content of triglyceride, adenosine and cordyceps polysaccharide to obtain a triglyceride distributed in the hydrophobic phase, 68.8% of triglyceride is distributed in the strong hydrophobic phase (phase I), the distribution coefficient of triglyceride in the weak hydrophobic phase is 0.61, adenosine and saccharides are distributed in the hydrophilic phase, 96.7% of adenosine is distributed in the weak hydrophilic phase (phase II), the selectivity coefficient of adenosine in the weak hydrophilic phase is 48, and the selectivity coefficient of adenosine remained in the coarse extracting solution is between the weak hydrophobic phase (insoluble phase) and the strong hydrophilic phase (phase III), 99.9% of the cordyceps polysaccharide is distributed in the IV phase, and the distribution coefficient of the cordyceps polysaccharide in the weak hydrophilic phase and the strong hydrophilic phase is 0.00085.
Example 9
Step (1): uniformly mixing soybean lecithin, camellia oil and n-hexane according to the mass ratio w/w/w =1:9:7 in a water bath at 45 ℃ (soybean lecithin is dissolved) and under magnetic stirring at 600rpm to obtain a camellia oil solution.
Step (2): taking 10g of tea seed cake powder, carrying out ultrasonic crushing for 30min at 750w by using 70% ethanol solution, carrying out water bath extraction for 2h at 60 ℃, centrifuging for 5min at 10000r/min, and taking supernatant to obtain tea seed cake extracting solution.
And (3): weighing 9.54g of ultrapure water, dissolving 2.7g of dipotassium hydrogen phosphate to obtain an inorganic salt solution, adding 5.76g of the camellia oil extract, adding 9g of the camellia oil solution, stirring uniformly, centrifuging for 3min at 9000r/min to form a four-liquid-phase system, respectively forming a strong hydrophobic phase, a weak hydrophilic phase, a weak hydrophobic phase and a strong hydrophilic phase from top to bottom, taking out each layer, respectively measuring the contents of triglyceride, phospholipid and total flavonoids to obtain triglyceride and phospholipid which are distributed in the hydrophobic phases, wherein 70.5% of triglyceride is distributed in the strong hydrophobic phase (phase I), the selectivity coefficient of triglyceride in the strong hydrophobic phase relative to the weak hydrophobic phase is 4.5, 72.6% of phospholipid is distributed in the weak hydrophobic phase (phase III), the distribution coefficient of phospholipid in the strong hydrophobic phase relative to the weak hydrophobic phase is 0.17, flavonoids are distributed in the hydrophilic phase, and 100% of total flavonoids are distributed in the weak hydrophilic phase (phase II), the insoluble extraction residue partitions at the interface between the weakly hydrophobic phase (phase iii) and the strongly hydrophilic phase (phase iv).
Example 10
Step (1): uniformly mixing soybean lecithin, soybean oil and n-hexane according to the mass ratio of w/w/w =1:9:3 in a water bath at 50 ℃ (soybean lecithin is dissolved) and under magnetic stirring at 500rpm to obtain a soybean oil solution.
Weighing 10.98g of water, adding 3.42g of dipotassium phosphate, dissolving and uniformly mixing to obtain a salt solution, adding 3.6g of absolute ethyl alcohol, adding 9g of the mixed soybean oil, uniformly stirring to obtain 28g of a four-phase system, adding 0.50g of cordyceps sinensis powder, extracting at 35 ℃ and 200rpm for 12h, centrifuging at 9000r/min for 5min to form a four-liquid-phase system, and taking out a strong hydrophobic phase, a weak hydrophilic phase and a strong hydrophilic phase from top to bottom in a layered manner. Preparing 7ml strong hydrophilic phase (IV phase) at the same ratio, mixing with the above four phases, centrifuging (changing volume ratio of each phase in the system), taking out each layer, the triglyceride, adenosine and saccharide contents were measured separately to obtain a distribution of triglyceride in the hydrophobic phase, a distribution coefficient of triglyceride in the strongly hydrophobic phase (phase I) of 1.86, distribution coefficients of adenosine and saccharide in the hydrophilic phase, 90.3% distribution of adenosine in the weakly hydrophilic phase (phase III), a selectivity coefficient of adenosine in the weakly hydrophilic phase with respect to the strongly hydrophilic phase of 39.8, 80.9% distribution of saccharide in the strongly hydrophilic phase (phase IV), a distribution coefficient of saccharide in the weakly hydrophilic phase with respect to the strongly hydrophilic phase of 0.39, and extraction residue mainly distributed at the interface of the weakly hydrophilic phase (phase III) and the strongly hydrophilic phase (phase IV).
Example 11
Step (1): uniformly mixing soybean lecithin, soybean oil and n-hexane according to the mass ratio of w/w/w =1:9:7.5 in a water bath at 50 ℃ (soybean lecithin is dissolved) and under magnetic stirring at 500rpm to obtain a soybean oil solution.
Weighing 11.16g of water, adding 2.34g of dipotassium phosphate, dissolving and uniformly mixing to obtain a salt solution, adding 4.5g of absolute ethyl alcohol, adding 9g of the mixed soybean oil, uniformly stirring to obtain 28g of a four-phase system, adding 0.50g of cordyceps sinensis powder, extracting at 35 ℃ and 200rpm for 12h, centrifuging at 9000r/min for 10min to respectively obtain extracting solutions of each layer, respectively preparing a strong hydrophobic phase, a weak hydrophilic phase and a strong hydrophilic phase from top to bottom, and taking out the extracting solutions in a layered manner. Preparing 10ml strong hydrophilic phase (IV phase) at the same ratio, mixing with the above four phases, centrifuging (changing volume ratio of each phase in the system), taking out each layer, the contents of triglyceride, adenosine and saccharide are respectively measured, the triglyceride is distributed in the hydrophobic phase, 79.9% of the triglycerides partition in the strongly hydrophobic phase (phase I), the partition coefficient of the triglycerides in the strongly hydrophobic phase with respect to the weakly hydrophobic phase is 1.4, the adenosine and the saccharides partition in the hydrophilic phase, where 88.9% of the adenosine was distributed in the weakly hydrophilic phase (phase III), the distribution coefficient of adenosine in the weakly hydrophilic phase versus the strongly hydrophilic phase was 9.37, 72.6% of the saccharides were distributed in the strongly hydrophilic phase (phase IV), the distribution coefficient of the saccharides in the weakly hydrophilic phase versus the strongly hydrophilic phase was 0.44, and the extraction residue was mainly distributed at the interface of the weakly hydrophilic phase (phase III) and the strongly hydrophilic phase (phase IV).
Example 12
Step (1): taking 10g Cordyceps powder, ultrasonically crushing with 70ml water at 250w for 30min, extracting in water bath at 80 deg.C for 3h, centrifuging at 10000r/min for 10min, and collecting supernatant to obtain Cordyceps powder extractive solution.
Weighing 11.16g of the cordyceps sinensis powder extracting solution, adding 3.24g of sodium sulfate, dissolving and uniformly mixing to obtain a salt solution, adding 3.6g of PEG600, adding 9g of soybean crude oil, stirring uniformly, finally adding 3.3g of monoglyceride laurate into the system, putting the monoglyceride laurate into a water bath at 55 ℃ for heating to dissolve the monoglyceride laurate, centrifuging for 3min at 9000r/min after the dissolution is finished to obtain a four-phase system, respectively preparing a strong hydrophobic phase, a weak hydrophilic phase and a strong hydrophilic phase from top to bottom, and taking out the materials in a layered manner. Preparing 6ml of strong hydrophilic phase (phase IV) according to the same proportion, uniformly mixing the strong hydrophilic phase (phase IV) with the four phases respectively taken out, performing centrifugal separation (namely changing the volume ratio of each phase in the system), taking out each layer, and respectively measuring the contents of phospholipid, adenosine and saccharide to obtain the phospholipid distributed in the hydrophobic phase, wherein 67.9% of the phospholipid is distributed in the weak hydrophobic phase (phase II), the distribution coefficient of the phospholipid in the weak hydrophobic phase is 0.157, the adenosine and the saccharide are distributed in the hydrophilic phase, wherein 93% of the adenosine is distributed in the weak hydrophilic phase (phase III), the distribution coefficient of the adenosine in the strong hydrophilic phase is 26.53, 70.8% of the saccharide is distributed in the strong hydrophilic phase (phase IV), and the distribution coefficient of the saccharide in the weak hydrophilic phase is 0.83.
Example 13
Step (1): mixing antarctic krill oil with n-hexane according to a mass-to-volume ratio w/v =1:9, stirring and uniformly mixing to obtain the antarctic krill oil solution.
Step (2): weighing 4.5g of water, adding 0.9g of dipotassium hydrogen phosphate to prepare an inorganic salt solution, adding 0.6g of absolute ethyl alcohol, adding 3g of the antarctic krill oil solution, shaking uniformly and fully, and centrifuging for 3min at 9000r/min to form a two-phase system (shown in figure 1 (a)); weighing 3.9g of water, adding 0.6g of dipotassium hydrogen phosphate to prepare an inorganic salt solution, adding 1.5g of absolute ethyl alcohol, adding 3g of the antarctic krill oil solution, shaking uniformly and fully, and centrifuging for 3min at 9000r/min to form a three-phase system (shown in figure 1 (b)); weighing 3.6g of water, adding 0.9g of dipotassium hydrogen phosphate to prepare an inorganic salt solution, adding 1.5g of absolute ethyl alcohol, adding 3g of the antarctic krill oil solution, shaking up the mixture by full shaking, and centrifuging the mixture for 3min at 9000r/min to form a four-phase system (figure 1 (c)). I.e. the system can only be in four phases at a range of ratios of the salt to the alcohol, as shown in figure 2.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for synchronously extracting and separating multiple components in raw materials by a four-liquid-phase system is characterized by comprising the following steps:
(1) mixing raw materials containing various components with different polarities with water, soluble salt and a hydrophilic solvent to prepare a four-liquid-phase system; the system contains hydrophobic substances and surfactants, wherein the surfactants are amphoteric surfactants and/or lipophilic nonionic surfactants;
(2) and centrifuging and/or standing the prepared four-liquid-phase system to form four liquid phases, respectively extracting each component in the raw materials into each layer of the strong hydrophobic layer, the weak hydrophilic layer and the strong hydrophilic layer according to the polarity difference, and distributing insoluble residues between phase interfaces.
2. The method according to claim 1, wherein a hydrophobic substance is further added to the system in the step (1), and the mass-to-volume ratio of the raw material to the hydrophobic substance is 0.1-4.0.
3. The method according to claim 1 or 2, wherein the hydrophobic substance is one or more of isooctane, n-hexane, ethyl acetate, chloroform, triglyceride, diglyceride, monoglyceride, astaxanthin, phospholipid, vitamin a, tocopherol, squalene, vitamin D, sterol, eicosapentaenoic acid, docosahexaenoic acid, oleic acid, and linoleic acid.
4. The method according to claim 1 or 2, characterized in that a surfactant is further added into the system in the step (1), and the mass ratio of the raw material to the surfactant is 0.1-3.0; the surfactant is one or more of phospholipid, phosphatidylcholine, monoglyceride laurate and monoglyceride stearate.
5. The method according to claim 1 or 2, wherein the hydrophilic solvent of step (1) is methanol, ethanol, isopropanol, PEG400, PEG600, [ BMIM [ ]]BF4、[BMIM]PF6One or a combination of several of them.
6. The method according to claim 5, wherein the soluble salt in step (1) is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate, sodium sulfate, sodium carbonate, ammonium sulfate and potassium chloride.
7. The method according to claim 1 or 2, wherein the mass ratio of the water, the soluble salt, the hydrophilic solvent and the raw materials in the step (2) is (0.25-0.70): (0.13-0.60): (0.15-0.60): (0.3-1.0).
8. The method according to claim 1 or 2, wherein the raw material in the step (1) is a pretreated raw material liquid; the mixing conditions in the step (1) are as follows: the temperature is 10-80 ℃, and the rotating speed is 100-1000 rpm.
9. The method of claim 8, wherein the pretreatment is one or more of hydrolysis, enzymolysis, ultrasonication, homogenization, and leaching.
10. The method of claim 9, wherein the raw material in step (1) comprises one or more of antarctic krill oil, fish oil, lard, beef tallow, soybean oil, camellia oil, olive oil, walnut oil, tea seed meal and cordyceps sinensis meal.
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