CN114644650A - Method for extracting high-purity sphingomyelin from egg yolk powder - Google Patents
Method for extracting high-purity sphingomyelin from egg yolk powder Download PDFInfo
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- CN114644650A CN114644650A CN202011523392.1A CN202011523392A CN114644650A CN 114644650 A CN114644650 A CN 114644650A CN 202011523392 A CN202011523392 A CN 202011523392A CN 114644650 A CN114644650 A CN 114644650A
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- sphingomyelin
- yolk powder
- egg yolk
- deoiled
- alcohol
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- 239000000843 powder Substances 0.000 title claims abstract description 74
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- 238000000034 method Methods 0.000 title claims abstract description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 79
- 238000000605 extraction Methods 0.000 claims abstract description 36
- 238000001035 drying Methods 0.000 claims abstract description 32
- 238000000926 separation method Methods 0.000 claims abstract description 25
- 239000003480 eluent Substances 0.000 claims abstract description 24
- 238000001914 filtration Methods 0.000 claims abstract description 24
- 239000000284 extract Substances 0.000 claims abstract description 19
- 239000000047 product Substances 0.000 claims abstract description 18
- 238000002156 mixing Methods 0.000 claims abstract description 15
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 15
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims abstract description 10
- 238000004108 freeze drying Methods 0.000 claims abstract description 10
- 229940067606 lecithin Drugs 0.000 claims abstract description 10
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 28
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- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
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- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 238000007670 refining Methods 0.000 claims description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
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- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 150000003904 phospholipids Chemical class 0.000 description 7
- UXTMROKLAAOEQO-UHFFFAOYSA-N chloroform;ethanol Chemical compound CCO.ClC(Cl)Cl UXTMROKLAAOEQO-UHFFFAOYSA-N 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- WTRDCBSIGOAXLO-UHFFFAOYSA-N chloroform;ethanol;hydrate Chemical compound O.CCO.ClC(Cl)Cl WTRDCBSIGOAXLO-UHFFFAOYSA-N 0.000 description 5
- 238000004042 decolorization Methods 0.000 description 5
- 239000000469 ethanolic extract Substances 0.000 description 5
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
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- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 238000002481 ethanol extraction Methods 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 238000000194 supercritical-fluid extraction Methods 0.000 description 3
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 2
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- CWRILEGKIAOYKP-SSDOTTSWSA-M [(2r)-3-acetyloxy-2-hydroxypropyl] 2-aminoethyl phosphate Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCCN CWRILEGKIAOYKP-SSDOTTSWSA-M 0.000 description 2
- LBVIBFFKPBBSCZ-UHFFFAOYSA-N butan-1-ol;octane Chemical group CCCCO.CCCCCCCC LBVIBFFKPBBSCZ-UHFFFAOYSA-N 0.000 description 2
- 229940106189 ceramide Drugs 0.000 description 2
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical group OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- CLUPOLFGIGLMIQ-UHFFFAOYSA-N heptane;propan-2-ol Chemical group CC(C)O.CCCCCCC CLUPOLFGIGLMIQ-UHFFFAOYSA-N 0.000 description 2
- 235000020256 human milk Nutrition 0.000 description 2
- 210000004251 human milk Anatomy 0.000 description 2
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- 238000011835 investigation Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 2
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 2
- 150000003905 phosphatidylinositols Chemical class 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- SUHOOTKUPISOBE-UHFFFAOYSA-N O-phosphoethanolamine Chemical compound NCCOP(O)(O)=O SUHOOTKUPISOBE-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- -1 and at the same time Inorganic materials 0.000 description 1
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- 239000012043 crude product Substances 0.000 description 1
- GTILCVOGKHZRBS-UHFFFAOYSA-N dichloromethane;ethanol;hydrate Chemical compound O.CCO.ClCCl GTILCVOGKHZRBS-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000003804 extraction from natural source Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000036449 good health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 235000021125 infant nutrition Nutrition 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 235000020978 long-chain polyunsaturated fatty acids Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
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- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
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- YHHSONZFOIEMCP-UHFFFAOYSA-O phosphocholine Chemical compound C[N+](C)(C)CCOP(O)(O)=O YHHSONZFOIEMCP-UHFFFAOYSA-O 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/113—Esters of phosphoric acids with unsaturated acyclic alcohols
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
- C07F9/103—Extraction or purification by physical or chemical treatment of natural phosphatides; Preparation of compositions containing phosphatides of unknown structure
Abstract
The invention relates to the field of natural medicine extraction and separation, and discloses a method for extracting high-purity sphingomyelin from egg yolk powder, which comprises the following steps: A. extracting deoiled yolk powder with alcohol, filtering to remove residue, mixing filtrates, concentrating, and drying to obtain deoiled yolk powder alcohol extract; B. completely dissolving the deoiled yolk powder alcohol extract, and performing silica gel column chromatography separation to leave a separated chromatographic column; C. eluting the separated chromatographic column by adopting an alkane-short chain alcohol system, merging the eluates, concentrating and drying to obtain a crude sphingomyelin product; D. and completely dissolving the crude sphingomyelin, performing secondary silica gel column chromatography, eluting by adopting an alkane-short chain alcohol-water system, collecting eluent, concentrating and freeze-drying to obtain a sphingomyelin product. The invention can obtain the high-purity sphingomyelin and the yolk lecithin, improves the utilization rate of raw materials and greatly reduces the production cost of the sphingomyelin.
Description
Technical Field
The invention relates to the field of natural medicine extraction and separation, and particularly relates to a method for extracting high-purity sphingomyelin from egg yolk powder.
Background
Sphingomyelin (SM), a sphingolipid composed of ceramide C-1 hydroxyl linked to phosphorylcholine (or phosphoethanolamine), is one of the bioactive lipids of human milk, and consists of sphingosine, fatty acid, phosphoric acid, and nitrogenous base. Sphingomyelin is the most abundant phospholipid in human milk fat, and accounts for 29.7% -45.5% of the total phospholipids. It has a high proportion in milk fat globule membrane, has many benefits for human health, and is a very important component in milk phospholipid fraction. It has been reported that sphingomyelin supplementation prevents disease and maintains good health. The phospholipid and the sphingomyelin have important functions in infant nutrition, and the phospholipid serving as a nutrition carrier of long-chain polyunsaturated fatty acid and sphingomyelin has a profound influence on the improvement of cognitive functions. Some researches show that sphingomyelin has a potential cancer inhibition effect, and two major metabolites, namely ceramide and sphingosine, of the sphingomyelin are involved in physiological activities such as cell growth, differentiation and apoptosis in organisms. Sphingomyelin has the above-mentioned benefits for human health, which makes sphingomyelin have a good application prospect.
The SM derived from natural sources is rich in animal brain, egg, etc., and has relatively low content in animal tissue. Therefore, the SM extraction from natural sources is carried out independently, the separation and purification cost is high, and the yield is too low. In addition, there are many impurity components in animal-derived phospholipids, and in the process of extracting natural egg yolk lecithin, for example, components such as Sphingomyelin (SM), Lysophosphatidylcholine (LPC), Lysophosphatidylethanolamine (LPE), Phosphatidylinositol (PI), etc. have similar polarities, and thus the separation difficulty is large. Therefore, the published technical reports are searched, and the fact that the related extraction process of the sphingomyelin with high purity is not reported, the SM content is only improved by aiming at the enrichment of SM-rich components, and the technical report of pure SM separation preparation is not reported from the aspects of pharmaceutic adjuvant and impurity separation is found, so in order to obtain the SM capable of meeting the medicinal requirement, the technical scheme of the invention starts from the SM-rich egg yolk powder, and obtains the SM with the purity of more than 98% through a series of technical means.
The natural fatty acid state of SM is better guaranteed by SM extracted from egg yolk powder and by SM obtained by enzymatic hydrolysis, which inevitably changes the natural state of SM due to the uncertainty of hydrolysis in enzymatic hydrolysis. As the content of the natural SM is very low, the SM used for the impurity inspection of the prior medicinal phospholipid products is obtained by adopting an enzyme hydrolysis process. However, the SM obtained by enzymatic hydrolysis is unreasonable to be used as a reference substance for SM detection in natural products, and a series of problems such as large fluctuation of detection results and inaccurate content measurement often occur. The optimal detection means is to adopt SM of natural source as a reference substance, but the content of SM of natural source is very low, the difficulty of extraction and separation is large, and a feasible obtaining method is lacked for a long time. Without a complete set of core technology, the SM is extracted only, and the extraction cost is high due to the large consumption of raw materials, and the adverse effect on the environment is caused. .
Disclosure of Invention
The invention aims to solve the problem that the prior art lacks a mature method for obtaining natural sphingomyelin.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for extracting high-purity sphingomyelin from egg yolk powder comprises the following steps:
A. extracting deoiled yolk powder with alcohol, filtering to remove residue, mixing filtrates, concentrating, and drying to obtain deoiled yolk powder alcohol extract;
B. completely dissolving the deoiled yolk powder alcohol extract, performing silica gel column chromatography, eluting and separating in a chloroform-ethanol system, and leaving a separated chromatographic column;
C. eluting the separated chromatographic column by adopting an alkane-short chain alcohol system, merging the eluates, concentrating and drying to obtain a crude sphingomyelin product;
D. and completely dissolving the crude sphingomyelin product, performing secondary silica gel column chromatography, eluting by adopting an alkane-short chain alcohol-water system, collecting eluent, concentrating and freeze-drying to obtain the sphingomyelin product.
Preferably, in step D, the alkane-short chain alcohol-water system comprises: alkane, short-chain alcohol and water in a volume ratio of 3: 5.85-5.95: 0.05 to 0.15.
Preferably, in the step D, the crude sphingomyelin is completely dissolved in chloroform, and the mass ratio of the crude sphingomyelin to silica gel is 1: 1 to 5 fillers, and the column pressure is 0.5 to 3.0 mPa.
Preferably, in step C, the alkane-short chain alcohol system comprises: alkane and short-chain alcohol according to the volume ratio of 3: 1-5 mixing.
Preferably, in the step B, the deoiled egg yolk powder alcohol extract is completely dissolved by using alkane, and the mass ratio of the deoiled egg yolk powder alcohol extract to silica gel is 1: 1-5 filling materials, and performing silica gel column chromatography separation, wherein the diameter-height ratio is 1: 1-1: 20, eluting with an alkane-short chain alcohol system comprising: alkane and short-chain alcohol according to the volume ratio of 3: 0.1 to 1.5, the column-passing pressure is-0.1 to 3.0mPa, and the column-passing temperature is 10 to 60 ℃.
In this step, the PC eluate is isolated by chromatography on a silica gel column. In the process of silica gel column chromatography elution, pure alkane is not good in elution effect alone, the polarity is increased by adding short-chain alcohol with proper proportion, but SM and LPC cannot be eluted, so that the eluent in one step needs to control the volume ratio of alkane to short-chain alcohol to be 3: 0.5-3: 1.5. Preferably, 3: 1 is the optimum condition for elution of PC.
Preferably, in step B, the deoiled yolk powder alcohol extract is adsorbed by alumina before being subjected to the silica gel column chromatography separation, specifically: and (2) completely dissolving the deoiled yolk powder alcohol extract by using short-chain alcohol, wherein the mass ratio of the deoiled yolk powder alcohol extract to the alumina is 1: stirring for 0.2-1, adsorbing for 1-10 h, filtering, concentrating, drying, dissolving for the second time, and performing silica gel column chromatography separation; collecting the chromatographic solution obtained by silica gel column chromatography, concentrating, decoloring with active carbon, filtering, concentrating, refining, and freeze-drying to obtain egg yolk lecithin product; the yolk lecithin content in the yolk lecithin product is more than 70%.
In the step, a large amount of Phosphatidylethanolamine (PE) is removed by adding alumina, stirring and adsorbing, and meanwhile, the effects of enriching Sphingomyelin (SM) and egg yolk lecithin (PC) are achieved, so that the egg yolk lecithin product with higher purity can be obtained.
Preferably, the alkane comprises one or more of chloroform, dichloromethane, n-heptane, and n-octane, and the short-chain alcohol comprises one or more of methanol, ethanol, n-butanol, and isopropanol.
In an alkane-short chain alcohol-water system or an alkane-short chain alcohol system used for elution, the longer the carbon chain of alcohol, the lower the polarity, the lower the selectivity of the composed eluent, which is easy to cause that a target sample cannot be eluted or cannot be completely eluted, and meanwhile, the alcohol with the overlong carbon chain is accompanied with strong pungent smell and is not suitable for use. The length of the short-chain alcohol is generally controlled within 5 carbons.
Preferably, in step a, the alcohol used for alcohol extraction includes one or more of methanol, ethanol and aqueous solution thereof; the mass part ratio of the deoiled yolk powder to the alcohol is 1: 5-15 times of extraction, 2-6 times of extraction, 1-8 h of extraction time and 15-50 ℃ of extraction temperature.
Preferably, in step a, the de-oiled egg yolk powder is obtained by de-oiling egg yolk powder, wherein the de-oiling comprises one of acetone de-oiling method and supercritical method; when the acetone deoiling method is applied, the mass ratio of the egg yolk powder to the acetone is 1: 1-10 times of deoiling, wherein the deoiling temperature is 20-45 ℃, and after deoiling, combining acetone insoluble substances, and drying to obtain the deoiled yolk powder; when the supercritical method is applied, the yolk powder is placed in an extraction kettle for continuous extraction, the temperature is kept at 25-45 ℃, and CO is kept2Controlling the flow rate at 50-120 kg/h, controlling the pressure of an extraction kettle at 10-25 Mpa, controlling the pressure of a separation kettle 1 at 5-20 Mpa, controlling the pressure of a separation kettle 2 at 5-15 Mpa, and continuing for 4-10 h to obtain the de-oiled yolk powder.
A sphingomyelin product obtained by the method for extracting high-purity sphingomyelin from egg yolk powder, wherein the purity of the sphingomyelin product is higher than 98%.
A product containing Sphingomyelin (SM) and egg yolk lecithin (PC) is obtained by the above method for extracting high purity sphingomyelin from egg yolk powder.
Compared with the prior art, the implementation of the invention has the following beneficial effects:
therefore, the technical scheme of the invention takes the SM-rich egg yolk powder as the starting material, and by adopting the technical scheme of the invention, not only can high-purity SM with the purity higher than 98% be obtained, but also egg yolk lecithin with the PC content higher than 70% can be obtained, so that the SM extraction cost is greatly reduced.
Drawings
FIG. 1 is a scheme of the sphingomyelin extraction process according to the technical solution of the present invention;
figure 2 is a graph comparing the effect of the second-order eluent on the purity of the crude SM and the finished SM.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail with reference to the accompanying drawings so that those skilled in the art can better understand the present invention and can implement the present invention, but the present invention is not limited by the illustrated examples. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
As shown in FIG. 1, egg yolk powder (1 kg) was deoiled by adding 10L of acetone, deoiled at 20 deg.C for 1 time, filtered, and dried to obtain deoiled egg yolk powder (732 g). And then 10L of 95% ethanol is used for carrying out ethanol extraction, the extraction temperature is 15 ℃, the extraction time is 1h, filter residues are filtered, repeated extraction is carried out for 2 times, filtrates are combined, and reduced pressure concentration is carried out to obtain 484g of the deoiled yolk powder ethanol extract. Adding ethanol to dissolve to prepare a raw material solution, adding 96.8g of alumina, stirring and adsorbing for 1 h. Filtering, concentrating, drying, dissolving with chloroform twice, filling 2.42kg silica gel for column chromatography (diameter-height ratio of 1: 1), at 10 deg.C, the first eluent is chloroform-ethanol (3: 0.1), the column pressure during elution is 1.0mPa, until the egg yolk lecithin (PC) is completely eluted, replacing with chloroform-ethanol (3: 2) for the second eluent, the column pressure during elution is 1.1mPa, eluting 10 column volumes, mixing eluates, concentrating, drying to obtain crude sphingomyelin 6.16g with sphingomyelin purity of 95.1%. Dissolving the crude sphingomyelin with chloroform to obtain a raw material solution, filling 20g of silica gel for column chromatography (diameter-height ratio of 1: 2), eluting with chloroform-ethanol-water (3: 1.85: 0.15), eluting at a column pressure of 0.5mPa until complete elution of sphingomyelin, mixing eluates containing sphingomyelin, concentrating, and drying to obtain 5.62g of sphingomyelin with a purity of 99.8%. Adding 0.3% (by solid content) of activated carbon into the PC eluent for decolorization and adsorption, filtering, concentrating, refining, and freeze-drying to obtain refined yolk lecithin with PC content of 74.6%.
Example 2
As shown in figure 1, 250g of egg yolk powder was taken, 1500ml of acetone was added for deoiling, deoiling was repeated 3 times at 30 ℃, filtered, and dried to obtain 162.85g of deoiled egg yolk powder. Extracting with 1950ml of 95% ethanol at 25 deg.C for 4 hr, filtering the residue, extracting repeatedly for 3 times, mixing the filtrates, concentrating, and drying to obtain 123.95g deoiled yolk powder ethanol extract. Adding methanol to dissolve to prepare a raw material solution, adding 62g of alumina, stirring and adsorbing for 3 hours. Then filtering, concentrating and drying, and dissolving with dichloromethane for the second time to obtain a concentrated solution. 375g of silica gel is filled for column chromatography (the diameter-height ratio is 1: 10), the column temperature is 25 ℃, the first eluent is dichloromethane-methanol (3: 0.5), the column pressure in the elution process is 3.0mPa until the yolk lecithin is completely eluted, the second eluent is dichloromethane-methanol (3: 3) is replaced, the column pressure in the elution process is 2.9mPa, 10 column volumes are eluted, the eluates are combined, concentrated and dried to obtain 2.01g of crude sphingomyelin, and the purity of the sphingomyelin is 88.4%. Dissolving the crude sphingomyelin with chloroform to obtain a raw material solution, filling 8g of silica gel for column chromatography (diameter-height ratio is 1: 5), eluting with dichloromethane-ethanol-water (3: 1.95: 0.05), the column pressure during elution is 1.5mPa until complete elution of sphingomyelin, combining the eluates containing sphingomyelin, concentrating and drying to obtain 1.74g of sphingomyelin with purity of 99.5%. Adding 0.1% (based on solid content) of active carbon into the PC eluent to perform decolorization and adsorption, filtering, concentrating, refining, and freeze-drying to obtain refined egg yolk lecithin with 76.1% of PC content after complete elution.
Example 3
As shown in FIG. 1, 500g of egg yolk powder was taken, 500ml of acetone was added to the egg yolk powder for deoiling, and deoiling was repeated 5 times at 45 ℃ and then filtered and dried to obtain 371g of deoiled egg yolk powder. Then using 1855ml 95% ethanol for ethanol extraction, the extraction temperature is 45 ℃, the extraction time is 8h, filtering the filter residue, repeatedly extracting for 6 times, combining the filtrates, concentrating and drying to obtain 292g of the deoiled yolk powder ethanol extract. Adding isopropanol to dissolve to prepare a raw material solution, adding 233.6g of alumina, stirring and adsorbing for 5 hours. Then filtering, concentrating and drying, dissolving with n-heptane twice to obtain concentrated solution, filling 292g silica gel for column chromatography (diameter-height ratio of 1: 15), the column temperature is 35 ℃, the first eluent is n-heptane-isopropanol (3: 1), the column pressure in the elution process is 1.6mPa until the egg yolk lecithin is completely eluted, replacing the second eluent with n-heptane-isopropanol (3: 4), the column pressure in the elution process is 1.8mPa, eluting 10 column volumes, combining the eluates, concentrating and drying to obtain 4.81g crude sphingomyelin, wherein the purity of sphingomyelin is 79.6%. Dissolving crude sphingomyelin with chloroform to obtain raw material solution, loading 25g silica gel, performing column chromatography (diameter-height ratio of 1: 3) with chloroform-ethanol-water (3: 1.90: 0.10) as eluent under 0.9mPa until sphingomyelin is completely eluted, mixing eluates containing sphingomyelin, concentrating, and drying to obtain sphingomyelin 3.66g with purity of 99.1%. Adding 0.5% (based on solid content) of active carbon into the PC eluent to perform decolorization and adsorption, filtering, concentrating, refining, and freeze-drying to obtain refined yolk lecithin with PC content of 72.8%.
Example 4
As shown in figure 1, 750g of yolk powder is taken and added into a supercritical extraction kettle for continuous extraction, the temperature of the extraction kettle is 45 ℃, and CO is added2The flow rate is 120kg/h, the pressure of the extraction kettle is 25MPa, the pressure of the separation kettle 1 is 20MPa, the pressure of the separation kettle 2 is 15MPa, and the operation lasts for 4 hours, so that 355.5g of the deoiled yolk powder is obtained. Extracting with 5330ml 95% ethanol at 50 deg.C for 8 hr, repeatedly extracting for 4 times, filtering to remove residue, concentrating, and drying to obtain 201.7g deoiled yolk powder ethanol extract. Adding n-butanol, dissolving to obtain raw material solution, adding 201.7g of alumina, stirring, and adsorbing for 8 hr. Then filtering, concentrating and drying, dissolving with n-octane for the second time, filling 605g of neutral alumina for column chromatography (the diameter-height ratio is 1: 20), the column temperature is 50 ℃, the first eluent is n-octane-n-butanol (3: 1.2) for elution, the column pressure in the elution process is 1.2mPa until the egg yolk lecithin is completely eluted, the n-octane-n-butanol (3: 5) in the second eluent is replaced, the column pressure in the elution process is 1.8mPa, the elution is carried out for 10 column volumes, the eluents are combined, concentrating and drying are carried out to obtain 7.64g of crude sphingomyelin, and the purity of sphingomyelin is 74.5%. Dissolving the crude sphingomyelin with chloroform to obtain a raw material solution, filling 7.64g of silica gel for column chromatography (diameter-height ratio is 1: 2), eluting with chloroform-ethanol-water (3: 1.85: 0.15), the column pressure during elution is 2.0mPa until sphingomyelin is completely eluted, combining the eluates containing sphingomyelin, concentrating and drying to obtain 5.62g of sphingomyelin with purity of 98.5%. Adding 0.7% (by solid content) of active carbon into PC eluate for decolorizing and adsorbing, eluting completely, filtering, concentrating, refining, and lyophilizing to obtain PC content 71.4%The refined egg yolk lecithin.
Example 5
As shown in figure 1, egg yolk powder 1kg is added into supercritical extraction kettle at 25 deg.C and CO for continuous extraction2The flow rate is 50kg/h, the pressure of the extraction kettle is 10MPa, the pressure of the separation kettle 1 is 5MPa, the pressure of the separation kettle 2 is 5MPa, and the flow lasts for 10 hours to obtain 676g of the deoiled yolk powder. Then using 6760ml 95% ethanol for ethanol extraction at 20 deg.C for 2h, filtering the residue, repeatedly extracting for 3 times, mixing the filtrates, concentrating, and drying to obtain 473g of deoiled egg yolk powder ethanol extract. Adding n-butanol to dissolve to obtain raw material solution, adding 330g of alumina, stirring and adsorbing for 10 h. Then filtering, concentrating and drying, dissolving with chloroform for two times, filling 2365g of neutral alumina for column chromatography (diameter-height ratio is 1: 8), the column temperature is 60 ℃, the first eluent is chloroform-n-butyl alcohol (3: 1.5), the column pressure in the elution process is 2.0mPa until the yolk lecithin is completely eluted, the chloroform-n-butyl alcohol (3: 5) in the second eluent is replaced, the column pressure in the elution process is 1.8mPa, eluting 10 column volumes, combining the eluates, concentrating and drying to obtain 11.26g of crude sphingomyelin, and the purity of sphingomyelin is 69.6%. Dissolving the crude sphingomyelin with chloroform to obtain a raw material solution, filling 66g of silica gel for column chromatography (diameter-height ratio is 1: 8), eluting with chloroform-ethanol-water (3: 1.90: 0.10), eluting at a column pressure of 3.0mPa until complete elution of sphingomyelin, mixing eluates containing sphingomyelin, concentrating, and drying to obtain 7.81g of sphingomyelin with a purity of 98.2%. Adding 0.7% (by solid content) of active carbon into the PC eluent for decolorization and adsorption, filtering, concentrating, refining, and freeze-drying to obtain refined egg yolk lecithin with PC content of 70.4%.
Comparative example 1
Adding yolk powder 2kg into supercritical extraction kettle at 35 deg.C and CO for continuous extraction2The flow rate is 80kg/h, the pressure of the extraction kettle is 15MPa, the pressure of the separation kettle 1 is 10MPa, the pressure of the separation kettle 2 is 8MPa, and the process lasts for 6 hours to obtain 1.2kg of the deoiled yolk powder. Extracting with 5.5L 95% ethanol at 35 deg.C for 4 hr, filtering, extracting the residue for 2 times, mixing filtrates, concentrating, and drying to obtain extract848g of oil egg yolk powder alcohol extract. Adding ethanol to dissolve to prepare a raw material solution, adding 169.6g of alumina, stirring and adsorbing for 1 h. Filtering, concentrating, drying, dissolving with chloroform twice, loading 4.8kg neutral alumina, performing column chromatography (diameter-height ratio of 1: 5), eluting with chloroform-ethanol (3: 1), eluting at column pressure of 1.8mPa until yolk lecithin is completely eluted, replacing chloroform-ethanol (3: 6) as the second eluent, eluting at column pressure of 2.4mPa for 10 column volumes, mixing eluates, concentrating, drying to obtain crude product 26.49g of sphingomyelin with purity of 63.9%. Dissolving the crude sphingomyelin with chloroform to obtain a raw material solution, filling 210g of silica gel for column chromatography (diameter-height ratio of 1: 15), eluting at a column pressure of 2.9mPa with chloroform-ethanol-water (3: 1.95: 0.05) until complete elution of sphingomyelin, mixing the eluates containing sphingomyelin, concentrating, and drying to obtain 16.86g of sphingomyelin with a purity of 96.7%. Adding 1.5% (by solid content) of activated carbon into the PC eluent for decolorization and adsorption, filtering, concentrating, refining, and freeze-drying to obtain refined egg yolk lecithin with PC content of 66.5%.
Comparative example 2
In order to fully evaluate the effect of the technology of the invention, the key steps of sphingomyelin elution are examined. In the process of extracting the sphingomyelin by adopting the yolk powder as a starting material, the first-stage elution and the second-stage elution are key steps for ensuring that the final purity of the sphingomyelin can reach more than 98 percent. When the de-oiled dried egg yolk alcohol extract is subjected to alumina column chromatography, most of SM and LPC are adsorbed in alumina, and at the same time, alumina also adsorbs a large amount of PC. In other words, after the deoiled yolk powder alcohol extract is subjected to alumina column chromatography, the main SM and LPC and a large amount of PC exist in the alumina column chromatography. Because of the very similar polarity of PC and SM, the first-stage elution requires the washing of a significant portion of the PC, and the complete removal of the first-stage PC directly affects the purity of the subsequent second column chromatography. Therefore, to ensure the purity of SM in the second elution step, all PC was eluted with chloroform-ethanol (3: 1), and then SM was eluted by increasing the ethanol ratio. The LPC is eluted along with the SM along with the increase of the polarity, so that the total mass and percentage of SM and LPC and the purity of SM after secondary column chromatography are taken as investigation indexes, the second-order elution proportion is investigated, the embodiment is carried out according to the technical scheme of example 1, and the second-order elution proportion is taken as a single factor for investigation. The ratio of chloroform to ethanol is respectively considered to be 3: 1,3: 2,3: 3,3: 4,3: 5,3: 6,3: 7 effect on final SM purity. Research results show that LPC is gradually eluted with the increase of polarity, the proportion of SM is gradually reduced, and after the percentage of SM in the first stage is less than 68%, sphingomyelin with the purity of more than 98% can not be obtained by secondary column chromatography, which is mainly because LPC and SM have very similar polarities, and high-purity SM is difficult to obtain. The results are shown in FIG. 2.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.
Claims (10)
1. A method for extracting high-purity sphingomyelin from egg yolk powder is characterized by comprising the following steps:
A. extracting deoiled yolk powder with alcohol, filtering to remove residue, mixing filtrates, concentrating, and drying to obtain deoiled yolk powder alcohol extract;
B. completely dissolving the deoiled yolk powder alcohol extract, and performing silica gel column chromatography separation to leave a separated chromatographic column;
C. eluting the separated chromatographic column by adopting an alkane-short chain alcohol system, merging the eluates, concentrating and drying to obtain a crude sphingomyelin product;
D. and completely dissolving the crude sphingomyelin, performing secondary silica gel column chromatography, eluting by adopting an alkane-short chain alcohol-water system, collecting eluent, concentrating and freeze-drying to obtain a sphingomyelin product.
2. The method for extracting high purity sphingomyelin from egg yolk powder according to claim 1, wherein in step D, said alkane-short chain alcohol-water system comprises: alkane, short-chain alcohol and water in a volume ratio of 3: 5.85-5.95: 0.05 to 0.15.
3. The method for extracting high-purity sphingomyelin from egg yolk powder according to claim 1, wherein in step D, the crude sphingomyelin is completely dissolved in chloroform, and the mass ratio of the crude sphingomyelin to silica gel is 1: 1 to 5 fillers, and the column pressure is 0.5 to 3.0 mPa.
4. The method for extracting high purity sphingomyelin from egg yolk powder according to claim 1, wherein in step C, said alkane-short chain alcohol system comprises: alkane and short-chain alcohol according to the volume ratio of 3: 1-5 mixing.
5. The method for extracting high-purity sphingomyelin from egg yolk powder according to claim 1, wherein in step B, said deoiled egg yolk powder alcohol extract is completely dissolved with alkane, and the mass ratio of said deoiled egg yolk powder alcohol extract to silica gel is 1: 1-5 filling materials, and performing silica gel column chromatography separation, wherein the diameter-height ratio is 1: 1-1: 20, eluting with an alkane-short chain alcohol system comprising: alkane and short-chain alcohol according to the volume ratio of 3: 0.1 to 1.5, the column passing pressure is-0.1 to 3.0mPa, and the column passing temperature is 10 to 60 ℃.
6. The method for extracting high-purity sphingomyelin from egg yolk powder according to claim 1, wherein in step B, the deoiled egg yolk powder alcohol extract is subjected to alumina adsorption before being subjected to the silica gel column chromatography separation, and specifically comprises: completely dissolving the deoiled yolk powder alcohol extract by using short-chain alcohol, and mixing the deoiled yolk powder alcohol extract with the alumina according to a mass ratio of 1: stirring for 0.2-1, adsorbing for 1-10 h, filtering, concentrating, drying, dissolving for the second time, and performing silica gel column chromatography separation; collecting the chromatographic solution obtained by silica gel column chromatography, concentrating, decoloring with active carbon, filtering, concentrating, refining, and freeze-drying to obtain egg yolk lecithin product; the yolk lecithin content in the yolk lecithin product is more than 70%.
7. The method for extracting high-purity sphingomyelin from egg yolk powder according to any one of claims 2 and 4 to 6, wherein the alkane comprises one or more of chloroform, dichloromethane, n-heptane and n-octane, and the short-chain alcohol comprises one or more of methanol, ethanol, n-butanol and isopropanol.
8. The method for extracting high-purity sphingomyelin from egg yolk powder according to claim 1, wherein in step a, the alcohol used for the alcohol extraction comprises one or more of methanol, ethanol and aqueous solutions thereof; the mass part ratio of the deoiled yolk powder to the alcohol is 1: 5-15 times of extraction, 2-6 times of extraction, 1-8 h of extraction time and 15-50 ℃ of extraction temperature.
9. The method for extracting high-purity sphingomyelin from egg yolk powder according to claim 1, wherein in step a, the de-oiled egg yolk powder is obtained by de-oiling egg yolk powder, wherein the de-oiling comprises one of acetone de-oiling and supercritical de-oiling; when the acetone deoiling method is applied, the mass ratio of the egg yolk powder to the acetone is 1: 1-10 times of deoiling, wherein the deoiling temperature is 20-45 ℃, and after deoiling, combining acetone insoluble substances, and drying to obtain the deoiled yolk powder; when the supercritical method is applied, the yolk powder is placed in an extraction kettle for continuous extraction, the temperature is kept at 25-45 ℃, and CO is kept2Controlling the flow rate at 50-120 kg/h, controlling the pressure of an extraction kettle at 10-25 Mpa, controlling the pressure of a separation kettle 1 at 5-20 Mpa, controlling the pressure of a separation kettle 2 at 5-15 Mpa, and continuing for 4-10 h to obtain the de-oiled yolk powder.
10. A sphingomyelin product obtained by the process for the extraction of high purity sphingomyelin from egg yolk powder according to claim 1 wherein said sphingomyelin product has a purity greater than 98%.
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